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US20240224964A9 - Correction of hepatosteatosis in humanized liver animals through restoration of il6/il6r/gp130 signaling in human hepatocytes - Google Patents

Correction of hepatosteatosis in humanized liver animals through restoration of il6/il6r/gp130 signaling in human hepatocytes Download PDF

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US20240224964A9
US20240224964A9 US18/478,273 US202318478273A US2024224964A9 US 20240224964 A9 US20240224964 A9 US 20240224964A9 US 202318478273 A US202318478273 A US 202318478273A US 2024224964 A9 US2024224964 A9 US 2024224964A9
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human
gene
human animal
inactivated endogenous
hepatocytes
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US20240130341A1 (en
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Joseph Zhe Li
Marisa Carbonaro
Gavin Thurston
Andrew J. Murphy
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/12—Animals modified by administration of exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes

Definitions

  • the non-human animal comprises an inactivated endogenous Rag1 gene and/or an inactivated endogenous Rag2 gene. In some such non-human animals, the non-human animal comprises an inactivated endogenous Rag2 gene. In some such non-human animals, the non-human animal comprises an inactivated endogenous Rag1 gene and an inactivated endogenous Rag2 gene. In some such non-human animals, the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene.
  • the non-human animal further comprises a GP130-activating ligand.
  • the GP130-activating ligand comprises human IL-6 or human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6).
  • the GP130-activating ligand comprises human IL-6 or human-IL-6R-compatible IL-6.
  • the GP130-activating ligand comprises human oncostatin-M (OSM) or human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM).
  • the genetically modified non-human animal comprises a humanized non-human animal SIRPA gene.
  • the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA, wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein.
  • the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter.
  • the genetically modified non-human animal is immunodeficient.
  • the non-human animal comprises an inactivated endogenous Il2rg gene.
  • the non-human animal comprises an inactivated endogenous Rag1 gene and/or an inactivated endogenous Rag2 gene.
  • the non-human animal comprises an inactivated endogenous Rag2 gene.
  • the non-human animal comprises an inactivated endogenous Rag1 gene and an inactivated endogenous Rag2 gene.
  • the genetically modified non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene.
  • the non-human animal comprises in its genome the GP130-activating ligand expression construct comprising a nucleic acid encoding the GP130-activating ligand operably linked to a promoter.
  • the GP130-activating ligand comprises human IL-6 or human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6).
  • the GP130-activating ligand comprises human IL-6 or human-IL-6R-compatible IL-6.
  • the GP130-activating ligand comprises human OSM or human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM).
  • the non-human animal is heterozygous for the humanized IL6 gene. In some such methods, the non-human animal is homozygous for the humanized IL6 gene. In some such methods, the non-human animal comprises the humanized IL6 gene in its germline. In some such methods, the non-human animal comprises a humanized non-human animal OSM gene comprising a human OSM nucleic acid encoding a human OSM protein. In some such methods, the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon. In some such methods, the human nucleic acid comprises a human OSM complementary DNA (cDNA).
  • cDNA human OSM complementary DNA
  • the GP130-activating ligands comprise: (1) human IL-6 or human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6); and (2) human OSM or human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM).
  • the modifying the non-human animal to comprise or express the GP130-activating ligand occurs prior to step (a).
  • the modifying the non-human animal to comprise or express the GP130-activating ligand occurs after step (a).
  • the non-human animal is a male. In some such methods, the non-human animal is a female. In some such methods, the non-human animal is a mammal. In some such methods, the mammal is a rodent. In some such methods, the rodent is a rat or a mouse. In some such methods, the rodent is the rat. In some such methods, the rodent is the mouse.
  • the human nucleic acid replaces a corresponding region of the non-human animal IL6 gene.
  • the human nucleic acid is inserted into the non-human animal IL6 gene.
  • the human nucleic acid is operably linked to an endogenous non-human animal IL6 promoter.
  • the non-human animal, cell, or genome is heterozygous for the humanized IL6 gene.
  • the non-human animal, cell, or genome is homozygous for the humanized IL6 gene.
  • a genetically modified non-human animal, non-human animal cell, or non-human animal genome comprising an inactivated endogenous Rag1 gene, an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, and a humanized OSM gene comprising a human OSM nucleic acid encoding a human OSM protein.
  • the human nucleic acid comprises a region of human OSM genomic sequence from the start codon to the stop codon.
  • the human nucleic acid comprises a human OSM complementary DNA (cDNA).
  • the human nucleic acid replaces a corresponding region of the non-human animal OSM gene.
  • the human nucleic acid is inserted into the non-human animal OSM gene.
  • the human nucleic acid is operably linked to an endogenous non-human animal OSM promoter.
  • the non-human animal is heterozygous for the humanized OSM gene.
  • the non-human animal is homozygous for the humanized OSM gene.
  • the non-human animal comprises the humanized OSM gene in its germline.
  • the animal, cell, or genome further comprises a humanized non-human animal SIRPA gene.
  • the humanized non-human animal SIRPA gene comprises a replacement of exons 2-4 of the non-human animal SIRPA gene with exons 2-4 of human SIRPA, wherein the humanized non-human animal SIRPA gene encodes a chimeric SIRPA protein comprising an extracellular portion of a human SIRPA protein and an intracellular portion of a non-human animal SIRPA protein.
  • the humanized non-human animal SIRPA gene is operably linked to an endogenous non-human animal SIRPA promoter.
  • the non-human animal is heterozygous for the humanized SIRPA gene. In some such animals, cells, or genomes, the non-human animal is homozygous for the humanized SIRPA gene. In some such animals, cells, or genomes, the non-human animal comprises the humanized SIRPA gene in its germline.
  • Some such methods further comprise modifying a non-human animal ES cell to generate the genetically modified non-human animal ES cell comprising the inactivated endogenous Rag2 gene, the inactivated endogenous Il2rg gene, the inactivated endogenous Fah gene, and the humanized OSM gene prior to step (a).
  • Some such methods comprise implanting and gestating a genetically modified non-human animal one-cell stage embryo comprising an inactivated endogenous Rag1 gene, an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, and a humanized OSM gene comprising a human OSM nucleic acid encoding a human OSM protein in a non-human animal surrogate mother, wherein the non-human animal surrogate mother produces an F0 progeny genetically modified non-human animal comprising the inactivated endogenous Rag1 gene, the inactivated endogenous Rag2 gene, the inactivated endogenous Il2rg gene, the inactivated endogenous Fah gene, and the humanized IL6 gene.
  • Some such methods further comprise modifying a non-human animal one-cell stage embryo to generate the genetically modified non-human animal one-cell stage embryo comprising the inactivated endogenous Rag1 gene, the inactivated endogenous Rag2 gene, the inactivated endogenous Il2rg gene, the inactivated endogenous Fah gene, and the humanized OSM gene prior to gestating the genetically modified non-human animal one-cell stage embryo in the non-human animal surrogate mother.
  • Some such methods comprise: (a) introducing a genetically modified non-human animal embryonic stem (ES) cell comprising an inactivated endogenous Rag1 gene, an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, a humanized IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein, and a humanized OSM gene comprising a human OSM nucleic acid encoding a human OSM protein into a non-human animal host embryo; and (b) implanting and gestating the non-human animal host embryo in a non-human animal surrogate mother, wherein the non-human animal surrogate mother produces an F0 progeny genetically modified non-human animal comprising the inactivated endogenous Rag1 gene, the inactivated endogenous Rag2 gene, the inactivated endogenous Il2rg gene, the inactivated endogenous Fah gene, the humanized IL6 gene, and the
  • Some such methods further comprise modifying a non-human animal ES cell to generate the genetically modified non-human animal ES cell comprising the inactivated endogenous Rag1 gene, the inactivated endogenous Rag2 gene, the inactivated endogenous Il2rg gene, the inactivated endogenous Fah gene, the humanized IL6 gene, and the humanized OSM gene prior to step (a).
  • Some such methods comprise implanting and gestating a genetically modified non-human animal one-cell stage embryo comprising an inactivated endogenous Rag1 gene, an inactivated endogenous Rag2 gene, an inactivated endogenous Il2rg gene, an inactivated endogenous Fah gene, a humanized IL6 gene comprising a human IL6 nucleic acid encoding a human IL-6 protein, and a humanized OSM gene comprising a human OSM nucleic acid encoding a human OSM protein in a non-human animal surrogate mother, wherein the non-human animal surrogate mother produces an F0 progeny genetically modified non-human animal comprising the inactivated endogenous Rag1 gene, the inactivated endogenous Rag2 gene, the inactivated endogenous Il2rg gene, the inactivated endogenous Fah gene, the humanized IL6 gene, and the humanized OSM gene.
  • Some such methods further comprise modifying a non-human animal one-cell stage embryo to generate the genetically modified non-human animal one-cell stage embryo comprising the inactivated endogenous Rag1 gene, the inactivated endogenous Rag2 gene, the inactivated endogenous Il2rg gene, the inactivated endogenous Fah gene, the humanized IL6 gene, and the humanized OSM gene prior to gestating the genetically modified non-human animal one-cell stage embryo in the non-human animal surrogate mother.
  • methods of making a non-human animal with a humanized liver comprise: (a) transplanting human hepatocytes or human hepatocyte progenitors into any of the above genetically modified non-human animals; (b) allowing the human hepatocytes or human hepatocyte progenitors to expand.
  • exogenous urokinase plasminogen activator or an exogenous nucleic acid encoding urokinase plasminogen activator is administered to the genetically modified non-human animal prior to step (a) to prime the liver for improved repopulation by human hepatocytes, optionally wherein the exogenous nucleic acid is an adenovirus or adeno-associated virus (AAV) encoding urokinase plasminogen activator.
  • AAV adeno-associated virus
  • exogenous herpes simplex virus type 1 thymidine kinase (HSVtk) or an exogenous nucleic acid encoding HSVtk is administered to the genetically modified non-human animal prior to step (a) to prime the liver for improved repopulation by human hepatocytes, optionally wherein the exogenous nucleic acid is an adenovirus or adeno-associated virus (AAV) encoding HSVtk.
  • the human hepatocytes or human hepatocyte progenitors are injected into the genetically modified non-human animal intrasplenically in step (a).
  • step (a) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency.
  • step (b) is done in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency.
  • nitisinone or any other compound that ameliorates toxicity caused by Fah deficiencies is administered to the non-human animals in step (b) in an on/off cycle to promote human hepatocyte repopulation.
  • the on/off cycle comprises about 5 to about 7 days off and about 3 days on.
  • the GP130-activating ligand comprises human IL-6 or human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6). In some such methods, the GP130-activating ligand comprises human IL-6 or human-IL-6R-compatible IL-6. In some such methods, the GP130-activating ligand comprises human OSM or human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM). In some such methods, the method further comprises administering one or more additional GP130-activating ligands or nucleic acids encoding the one or more additional GP130-activating ligand to the non-human animal.
  • the promoter is a tissue-specific promoter, optionally wherein the tissue-specific promoter is a liver-specific promoter or a muscle-specific promoter, optionally wherein the muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7).
  • the promoter is a constitutive promoter.
  • the vector is a viral vector.
  • the viral vector is a lentivirus vector or an adeno-associated virus (AAV) vector.
  • the viral vector is the AAV vector, optionally wherein the AAV vector is a recombinant AAV9 vector.
  • the non-human animal is immunodeficient. In some such methods, the non-human animal comprises an inactivated endogenous Il2rg gene. In some such methods, the non-human animal comprises an inactivated endogenous Rag1 gene and/or an inactivated endogenous Rag2 gene. In some such methods, the non-human animal comprises an inactivated endogenous Rag2 gene. In some such methods, the non-human animal comprises an inactivated endogenous Rag1 gene and an inactivated endogenous Rag2 gene. In some such methods, the non-human animal comprises an inactivated endogenous Rag2 gene and an inactivated endogenous Il2rg gene.
  • FIGS. 1 A- 1 C show abnormal lipid accumulation in humanized livers of mice and rats.
  • H&E, FAH, hASGR1 and HSD17B13 IHC and Oil Red O staining show lipid accumulation in both humanized mouse ( FIG. 1 A ) and rat ( FIG. 1 B ) livers, collected at 12 weeks post-transplant and 7 months post-transplant, respectively.
  • FIG. 1 A shows abnormal lipid accumulation in humanized livers of mice and rats.
  • H&E, FAH, hASGR1 and HSD17B13 IHC and Oil Red O staining show lipid accumulation in both humanized mouse ( FIG. 1 A ) and rat ( FIG. 1 B ) livers, collected at 12 weeks post-transplant and 7 months post-transplant, respectively.
  • FIG. 1 A humanized mouse
  • rat FIG. 1 B
  • FIGS. 2 C and 2 E upper panels show H&E, FAH IHC, hASGR1 IHC, and FLAG (IHC or miRNAscope) staining of FSRG mouse ( FIG. 2 C ) or FRG rat ( FIG. 2 E ) livers engrafted with mIL-6R- or rIL-6R-expressing PHH (FLAG+ hepatocytes) or non-transduced PHH (FLAG-hepatocytes).
  • FIG. 2 C and 2 E upper panels show H&E, FAH IHC, hASGR1 IHC, and FLAG (IHC or miRNAscope) staining of FSRG mouse ( FIG. 2 C ) or FRG rat ( FIG. 2 E ) livers engrafted with mIL-6R- or rIL-6R-expressing PHH (FLAG+ hepatocytes) or non-transduced PHH (FLAG-hepatocytes).
  • FIG. 1 2 C lower panels show H&E and hASGR1-IHC/FLAG-RNAScope double staining of FSRG mouse livers engrafted with PHH expressing mIL6R, collected after 14 weeks.
  • mIL6R-expressing human hepatocytes Hu-mIL6R
  • Non-transduced human hepatocytes Hu
  • Non-engrafted regions Mo
  • Hu-GP130* hepatocytes were detected by hASGR1 IHC and GFP-RNAScope.
  • Non-transduced human hepatocytes (Hu) were positive for hASGR1, but not GFP.
  • Non-engrafted regions (Mo) are negative for both.
  • Experiment performed twice with 2 hepatocyte donors, n 3 per cohort.
  • FIG. 4 B shows H&E and FAH IHC from PHH-engrafted livers of IL-6 m/m , IL-6 h/m , or IL-6 h/h mice. Quantification shows the % Fatty Area (negative H&E staining) and % FAH+ staining (Mean ⁇ SD, 3 liver sections/group; **p ⁇ 0.01 one-way ANOVA).
  • FIGS. 5 A- 5 B show human IL-6 over-expression corrects lipid droplet accumulation in humanized liver mice and rats.
  • FIGS. 5 A and 5 B show H&E, FAH IHC, and Oil Red O staining on livers from AAV9-hIL-6 versus PBS treated humanized liver mice 8 weeks after PHH transplantation ( FIG. 5 A ) or humanized liver rats 12 weeks after PHH transplantation ( FIG. 5 B ), both collected 4 weeks after AAV dosing.
  • FIG. 10 B shows human IL-6 was detected (predominately in CD68 positive cells) in the livers of FSRG-IL-6 HumIn(het) but not FSRG-IL-6 WT humanized liver mice after 2 hr LPS stimulation, as shown by H&E, FAH IHC, hIL-6, mIL-6, and mCD68 RNAscope staining.
  • FIGS. 13 A- 13 B show IL-6 expression and signaling in the livers of FSRG-hIL-6 mice.
  • FIG. 13 A shows hCRP and hAlb in the serum of PHH-engrafted FSRG-IL-6 m/m , IL-6 h/m , or IL-6 h/h mice.
  • FIG. 13 B shows H&E, FAH IHC, hIL-6, mIL-6, and mCD68 RNAscope staining of livers from either FSRG-IL-6 h/m or FSRG-IL-6 m/m mice after 2 hours LPS stimulation.
  • FIG. 14 D shows H&E staining and FAH IHC on liver sections from AAV9-hOSM and PBS treated mice, 3 weeks after AAV dosing. Quantification of the % fatty area (negative H&E staining) and FAH staining shows mean ⁇ SD (each dot represents one mouse, 2 liver lobes/mouse were analyzed). *p ⁇ 0.05, Unpaired t test.
  • FIGS. 15 A- 15 D show that engraftment of human immune cells led to hIL-6 expression in HIS-HuHEP mice.
  • FIG. 15 A shows serum levels of human albumin, human IL-6, and human CRP, and % hCD45+ cells in the blood confirm dual-humanization and intact IL-6 signaling in HIS-HuHEP mice.
  • FIG. 15 B shows single and double-IHC for hCD45 and hCD68 in HIS-HuHEP mouse livers.
  • FIG. 15 C shows serum ELISAs that demonstrate a complete absence of hCRP in anti-CSF1R treated mice, despite high human albumin levels.
  • FIG. 15 A shows serum levels of human albumin, human IL-6, and human CRP, and % hCD45+ cells in the blood confirm dual-humanization and intact IL-6 signaling in HIS-HuHEP mice.
  • FIG. 15 B shows single and double-IHC for hCD45 and hCD68 in HIS-HuHEP
  • protein polypeptide
  • polypeptide include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids.
  • the terms also include polymers that have been modified, such as polypeptides having modified peptide backbones.
  • domain refers to any part of a protein or polypeptide having a particular function or structure.
  • nucleic acid that has been introduced into a cell such that the nucleotide sequence integrates into the genome of the cell. Any protocol may be used for the stable incorporation of a nucleic acid into the genome of a cell.
  • viral vector refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle.
  • the vector and/or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either ex vivo or in vivo. Numerous forms of viral vectors are known.
  • isolated may include proteins, nucleic acids, or cells that have been separated or purified from most other cellular components or organism components with which they are naturally accompanied (e.g., but not limited to, other cellular proteins, nucleic acids, or cellular or extracellular components).
  • wild type includes entities having a structure and/or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).
  • endogenous sequence refers to a nucleic acid sequence that occurs naturally within a cell or animal.
  • an endogenous IL6 sequence of an animal refers to a native IL6 sequence that naturally occurs at the IL6 locus in the animal.
  • inducible promoters include, for example, chemically regulated promoters and physically regulated promoters.
  • Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal-regulated promoters (e.g., a metalloprotein promoter).
  • Physically regulated promoters include, for example temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e
  • any suitable immunodeficient non-human animal can be used. See, e.g., Weber et al. (2009) Liver Transplantation 15:7-14, herein incorporated by reference in its entirety for all purposes.
  • Such non-human animals can be immunocompromised such that T cells and B cells do not develop.
  • such immunodeficient non-human animals can lack functional T cells, B cells, and/or natural killer (NK) cells.
  • Immunodeficient non-human animals refer to non-human animals lacking in at least one essential function of the immune system.
  • an immunodeficient non-human animal is one lacking specific components of the immune system or lacking function of specific components of the immune system (such as, for example, B cells, T cells, or NK cells).
  • genetically modified non-human animals for xenotransplantation of hepatocytes have the following genes inactivated (i.e., knocked out): Fah (encodes fumarylacetoacetase); Rag1 (encodes V(D)J recombination-activating protein 1); Rag2 (encodes V(D)J recombination-activating protein 2); and Il2rg (encodes interleukin 2 receptor subunit gamma).
  • Fah encodes fumarylacetoacetase
  • Rag1 encodes V(D)J recombination-activating protein 1
  • Rag2 encodes V(D)J recombination-activating protein 2
  • Il2rg encodes interleukin 2 receptor subunit gamma
  • Also provided are genetically modified non-human animal cells or genomes having the following genes inactivated (i.e., knocked out): Fah; Rag1; Rag2; and Il2rg.
  • RAG1 mediates the DNA-binding to the conserved recombination signal sequences (RSS) and catalyzes the DNA cleavage activities by introducing a double-strand break between the RSS and the adjacent coding segment.
  • RAG2 is not a catalytic component but is required for all known catalytic activities.
  • RAG1 and RAG2 are essential to the generation of mature B cells and T cells, two types of lymphocytes that are crucial components of the adaptive immune system.
  • An inactivated endogenous Rag2 gene is a Rag2 gene that does not produce a RAG2 protein or does not produce a functional RAG2 protein.
  • the non-human animal (or cell or genome) can comprise the inactivated Rag2 gene in its germline.
  • the non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Rag2 gene.
  • an inactivated endogenous Rag2 gene can comprise an insertion, a deletion, or one or more point mutations in the endogenous Rag2 gene resulting in loss of expression of functional RAG2 protein.
  • Mouse Il2rg maps to X D; X 43.9 cM on chromosome X (NCBI RefSeq Gene ID 16186; Assembly GRCm39 (GCF_000001635.27); location NC_000086.8 (100307991 . . . 100311861, complement).
  • Reference to the mouse Il2rg gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical, wild type mouse IL2rG protein has been assigned UniProt accession number P34902 and NCBI Accession No. NP_038591.1.
  • Reference to mouse IL2RG proteins includes canonical, wild type forms as well as all allelic forms and isoforms.
  • an inactivated endogenous Il2rg gene can be one in which the start codon of the endogenous Il2rg gene has been deleted or has been disrupted or mutated such that the start codon is no longer functional.
  • the start codon can be disrupted by a deletion or insertion within the start codon.
  • the start codon can be mutated by, for example, by a substitution of one or more nucleotides.
  • a 3′ fragment of the Il2rg gene can be deleted or disrupted (e.g., including the stop codon).
  • an internal fragment of the Il2rg gene i.e., a fragment from the middle of the Il2rg gene
  • all of the coding sequence in the endogenous Il2rg gene is deleted or disrupted.
  • Fumarylacetoacetase (also known as FAH, FAA, beta-diketonase, or fumarylacetoacetate hydrolase) is encoded by the Fah gene (also known as fumarylacetoacetate hydrolase).
  • Fah is an enzyme required in the last step of the tyrosine catabolic pathway, which hydrolyzes fumarylacetoacetate into fumarate and acetoacetate.
  • a deficiency in Fah leads to the accumulation of toxic metabolites, including fumarylacetoacetate and maleylacetoacetate.
  • the altered Fah gene that causes this condition produces an unstable or inactive enzyme, which results in reduced or absent fumarylacetoacetate hydrolase activity. Without sufficient fumarylacetoacetate hydrolase activity, tyrosine and its byproducts are not properly broken down. As a result, fumarylacetoacetate accumulates in the liver and kidneys. Elevated levels of fumarylacetoacetate are thought to be toxic to cells and accumulation of this substance likely causes the liver and kidney problems and other features that are characteristic of tyrosinemia type I.
  • mRNA encoding the canonical isoform is assigned NCBI Accession No. NM_010176.4.
  • Reference to the mouse Fah mRNA (cDNA) and coding sequence includes the canonical, wild type forms as well as all allelic forms and isoforms.
  • An inactivated endogenous Fah gene is a Fah gene that does not produce a FAH protein or does not produce a functional FAH protein.
  • the non-human animal (or cell or genome) can comprise the inactivated Fah gene in its germline.
  • the non-human animal (or cell or genome) can be homozygous for an inactivating mutation in the Fah gene.
  • an inactivated endogenous Fah gene can comprise an insertion, a deletion, or one or more point mutations in the endogenous Fah gene resulting in loss of expression of functional FAH protein.
  • the start codon can be disrupted by a deletion or insertion within the start codon.
  • the start codon can be mutated by, for example, by a substitution of one or more nucleotides.
  • a 3′ fragment of the Fah gene can be deleted or disrupted (e.g., including the stop codon).
  • an internal fragment of the Fah gene i.e., a fragment from the middle of the Fah gene
  • all of the coding sequence in the endogenous Fah gene is deleted or disrupted.
  • SIRPA also known as BIT, MFR, MYD 1, PTPNS1, SHPS1, SIRP ⁇ , and SIRP
  • tyrosine-protein phosphatase non-receptor type substrate 1 also known as brain Ig-like molecule with tyrosine-based activation motifs (Bit), CD172 antigen-like family member A, CD172a, inhibitory receptor SHPS-1, macrophage fusion receptor, MyD-1 antigen, SIRPA, SIRP ⁇ , and signal-regulatory protein alpha
  • It acts as docking protein and induces translocation of PTPN6, PTPN11 and other binding partners from the cytosol to the plasma membrane.
  • Mouse Sirpa maps to 2 F1; 2 63.19 cM on chromosome 2 (NCBI RefSeq Gene ID 19261; Assembly GRCm39 (GCF_000001635.27); location NC_000068.8 (129432962 . . . 129474148).
  • Reference to the mouse Sirpa gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical mouse SIRPA protein has been assigned UniProt accession number P97797 and NCBI Accession Nos. NP_001277948.1 and NP_001277949.1.
  • Reference to mouse SIRPA proteins includes canonical forms as well as all allelic forms and isoforms.
  • mRNAs encoding the canonical isoform are assigned NCBI Accession Nos. NM_001291019.1 and NM_001291020.1.
  • Reference to the mouse Sirpa mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • Human SIRPA maps to 20p13 on chromosome 20 (NCBI RefSeq Gene ID 140885; Assembly GRCh38.p14 (GCF_000001405.40); location NC_000020.11 (1894167 . . . 1940592).
  • Reference to the human SIRPA gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical human SIRPA protein has been assigned UniProt accession number P78324 and NCBI Accession Nos. NP_001035111.1, NP_001035112.1, and NP_542970.1.
  • Reference to human SIRPA proteins includes the canonical form as well as all allelic forms and isoforms.
  • mRNAs encoding the canonical isoform are assigned NCBI Accession Nos. NM_001040022.1, NM_001040023.1, and NM_080792.2.
  • Reference to the human SIRPA mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • the human SIRPA nucleic acid can be inserted into the non-human animal Sirpa genomic locus, or it can replace a corresponding region of the non-human animal Sirpa locus (e.g., a region of a human SIRPA gene comprising exons 2-4 can replace exons 2-4 of the non-human animal Sirpa gene).
  • the humanized SIRPA gene (or the human SIRPA nucleic acid) can be operably linked to the endogenous non-human animal Sirpa promoter. In other words, expression of the humanized SIRPA gene can be driven by the endogenous non-human animal Sirpa promoter.
  • Some genetically modified non-human animals described herein further comprise a humanized IL6 gene.
  • such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and a humanized IL6 gene.
  • such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA and IL6 genes.
  • such genetically modified non-human animals can comprise inactivated Fah, Rag1, Rag2, and Il2rg genes and a humanized IL6 gene.
  • such genetically modified non-human animals can comprise inactivated Fah, Rag1, Rag2, and Il2rg genes and humanized SIRPA and IL6 genes.
  • such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized IL6 gene.
  • the non-human animal is a mouse.
  • the non-human animal is a rat.
  • IL6 also known as IL-6 and IFNB2 encodes interleukin-6 (also known as IL-6, IL6, B-cell stimulatory factor 2 (BSF-2), CTL differentiation factor (CDF), hybridoma growth factor, and interferon beta-2 (IFN-beta-2).
  • IL-6 is a cytokine with a wide variety of biological functions in immunity, tissue regeneration, and metabolism. It binds to IL-6R, then the complex associates with the signaling subunit IL6ST/gp130 to trigger the intracellular IL6-signaling pathway.
  • Rat Il6 maps to 4q11 on chromosome 4 (NCBI RefSeq Gene ID 24498; Assembly mRatBN7.2 (GCF_015227675.2); location NC_051339.1 (5214602 . . . 5219178, complement).
  • Reference to the rat Il6 gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical rat IL-6 protein has been assigned UniProt accession number P20607 and NCBI Accession No. NP_036721.1.
  • Reference to rat IL-6 proteins includes canonical forms as well as all allelic forms and isoforms.
  • An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_012589.2.
  • Reference to the rat Il6 mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • NM_000600.4 A coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS5375.1 (SEQ ID NO: 44).
  • Reference to the human IL6 mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • non-human animals with humanized IL6 genes are provided, e.g., in U.S. Pat. No. 9,622,460, herein incorporated by reference in its entirety for all purposes.
  • the non-human animal (or cell or genome) can be, in some cases, homozygous for the humanized IL6 gene. In other cases, the animal (or cell or genome) can be heterozygous for the humanized IL6 gene.
  • the non-human animal can comprise the humanized IL6 gene in its germline.
  • the humanized IL6 gene can comprise a human IL6 nucleic acid encoding a human IL-6 protein (e.g., a fully human IL-6 protein).
  • the human IL6 nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human IL6 gene from the start codon to the stop codon, or can comprise a human IL6 complementary DNA (cDNA).
  • the human IL6 nucleic acid can be inserted into the non-human animal 116 genomic locus, or it can replace a corresponding region of the non-human animal 116 locus (e.g., a region of a human IL6 gene from the start codon to the stop codon can replace a region of the non-human animal 116 gene from the start codon to the stop codon).
  • such genetically modified non-human animals can be immunodeficient non-human animals comprising a humanized OSM gene.
  • such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized IL6 and OSM genes.
  • such genetically modified non-human animals can comprise inactivated Fah, Rag2, and Il2rg genes and humanized SIRPA, IL6, and OSM genes.
  • such genetically modified non-human animals can comprise inactivated Fah, Rag1, Rag2, and Il2rg genes and humanized IL6 and OSM genes.
  • OSM encodes oncostatin-M (also known as OSM).
  • OSM is a pleiotropic cytokine that belongs to the interleukin 6 group of cytokines. Of these cytokines, it most closely resembles leukemia inhibitory factor (LIF) in both structure and function. It is important in liver development, hematopoiesis, inflammation and possibly CNS development. It is also associated with bone formation and destruction.
  • LIF leukemia inhibitory factor
  • OSM signals through cell surface receptors that contain the protein GP130.
  • the type I receptor is composed of GP130 and LIFR
  • the type II receptor is composed of GP130 and OSMR.
  • Mouse Osm maps to 11 A1; 11 2.94 cM on chromosome 11 (NCBI RefSeq Gene ID 18413; Assembly GRCm39 (GCF_000001635.27); location NC_000077.7 (4186785 . . . 4191026).
  • Reference to the mouse Osm gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical mouse OSM protein has been assigned UniProt accession number P53347 and NCBI Accession No. NP_001013383.1.
  • Reference to mouse OSM proteins includes canonical forms as well as all allelic forms and isoforms.
  • mRNAs (cDNAs) encoding the canonical isoform are assigned NCBI Accession No. NM_001013365.3.
  • Reference to the mouse Osm mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • Rat Osm maps to 14q21 on chromosome 14 (NCBI RefSeq Gene ID 289747; Assembly mRatBN7.2 (GCF_015227675.2); location NC_051349.1 (79103638 . . . 79108500).
  • Reference to the rat Osm gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical rat OSM protein has been assigned UniProt accession number Q65Z15 and NCBI Accession No. NP_001006962.1.
  • Reference to rat OSM proteins includes canonical forms as well as all allelic forms and isoforms.
  • An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_001006961.2.
  • Reference to the rat Osm mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • Human OSM maps to 22q12.2 on chromosome 22 (NCBI RefSeq Gene ID 5008; Assembly GRCh38.p14 (GCF_000001405.40); location NC_000022.11 (30262829 . . . 30266851, complement).
  • Reference to the human OSM gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical human OSM protein has been assigned UniProt accession number P13725 and NCBI Accession No. NP_065391.1 (SEQ ID NO: 51).
  • Reference to human OSM proteins includes the canonical form as well as all allelic forms and isoforms.
  • An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No.
  • NM_020530.6 A coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS13873.1 (SEQ ID NO: 50).
  • Reference to the human OSM mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • Some genetically modified non-human animals described herein further comprise a humanized Growth Hormone gene. Similar to IL6, mGH shows species specificities in receptor binding, and administration of human Growth Hormone (GH) in humanized liver mice could correct fatty liver in humanized liver mice. Tateno et al. (2011) Endocrinology 152:1479-1491, herein incorporated by reference in its entirety for all purposes.
  • GH Growth Hormone
  • non-human animals can be male or female.
  • Non-human animal cells disclosed herein can be any type of undifferentiated or differentiated state.
  • a non-human animal cell can be a totipotent cell, a pluripotent cell (e.g., mouse or rat pluripotent cell such as a mouse or rat embryonic stem (ES) cell), or a non-pluripotent cell.
  • Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that possess the ability to develop into more than one differentiated cell types.
  • pluripotent and/or totipotent cells can be, for example, ES cells or ES-like cells, such as an induced pluripotent stem (iPS) cells.
  • ES cells include embryo-derived totipotent or pluripotent cells that are capable of contributing to any tissue of the developing embryo upon introduction into an embryo.
  • ES cells can be derived from the inner cell mass of a blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).
  • the cells provided herein can also be germ cells (e.g., sperm or oocytes).
  • the cells can be mitotically competent cells or mitotically inactive cells, meiotically competent cells or meiotically-inactive cells.
  • the cells can also be primary somatic cells or cells that are not a primary somatic cell. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell.
  • the cells can be liver cells (e.g., hepatocytes).
  • Suitable cells provided herein also include primary cells.
  • Primary cells include cells or cultures of cells that have been isolated directly from an organism, organ, or tissue.
  • Primary cells include cells that are neither transformed nor immortal. They include any cell obtained from an organism, organ, or tissue which was not previously passed in tissue culture or has been previously passed in tissue culture but is incapable of being indefinitely passed in tissue culture.
  • Such cells can be isolated by conventional techniques and include, for example, liver cells (e.g., hepatocytes).
  • Immortalized cells include cells from a multicellular organism that would normally not proliferate indefinitely but, due to mutation or alteration, have evaded normal cellular senescence and instead can keep undergoing division. Such mutations or alterations can occur naturally or be intentionally induced. Numerous types of immortalized cells are well known. Immortalized or primary cells include cells that are typically used for culturing or for expressing recombinant genes or proteins.
  • the cells provided herein also include one-cell stage embryos (i.e., fertilized oocytes or zygotes). Such one-cell stage embryos can be from any genetic background, can be fresh or frozen, and can be derived from natural breeding or in vitro fertilization.
  • the cells provided herein can be normal, healthy cells, or can be diseased or mutant-bearing cells.
  • the non-human animal can be a eukaryote, which includes, for example, animals and mammals.
  • the term “animal” includes any member of the animal kingdom, including, for example, mammals, fishes, reptiles, amphibians, and birds.
  • a mammal can be, for example, a pig, a rodent, a rat, or a mouse.
  • Other mammals include, for example, non-human primates, cats, dogs, rabbits, cows, sheep, goats, pigs, and boars, and so forth.
  • Birds include, for example, chickens, turkeys, ostrich, geese, ducks, and so forth.
  • the term “non-human” excludes humans.
  • the non-human animals can be from any genetic background.
  • suitable mice, mouse cells, or mouse genomes can be from a 129 strain, a C57BL/6 strain, a mix of 129 and C57BL/6, a BALB/c strain, or a Swiss Webster strain.
  • 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 12951/SV, 12951/Svlm), 129S2, 129S4, 129S5, 12959/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, and 129T2. See, e.g., Festing et al. (1999) Mamm.
  • C57BL strains include C57BL/A, C57BL/An, C57BL/GrFa, C57BL/Kal_wN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.
  • Suitable mice can also be from a mix of an aforementioned 129 strain and an aforementioned C57BL/6 strain (e.g., 50% 129 and 50% C57BL/6).
  • suitable mice can be from a mix of aforementioned 129 strains or a mix of aforementioned BL/6 strains (e.g., the 129S6 (129/SvEvTac) strain).
  • Rats or rat cells or rat genomes can be from any rat strain, including, for example, an ACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, a LEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer rat strain such as Fisher F344 or Fisher F6.
  • Rats, rat cells, or rat genomes can also be obtained from a strain derived from a mix of two or more strains recited above.
  • a suitable rat can be from a DA strain or an ACI strain.
  • the ACI rat strain is characterized as having black agouti, with white belly and feet and an RT1 av1 haplotype.
  • Such strains are available from a variety of sources including Harlan Laboratories.
  • the Dark Agouti (DA) rat strain is characterized as having an agouti coat and an RT1 av1 haplotype.
  • Such rats are available from a variety of sources including Charles River and Harlan Laboratories.
  • Some suitable rats, rat cells, and rat genomes can be from an inbred rat strain. See, e.g., US 2014/0235933, herein incorporated by reference in its entirety for all purposes.
  • the non-human animals can exhibit one or more of the following (e.g., in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency and/or in the absence of transplantation of hepatocytes with a functional copy of Fah): hypertyrosinemia; liver fibrosis; cirrhosis; liver failure; and renal tubular damage or dysfunction.
  • hypertyrosinemia e.g., in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency and/or in the absence of transplantation of hepatocytes with a functional copy of Fah
  • hypertyrosinemia e.g., in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency and/or in the absence of transplantation of hepatocytes with a functional copy of Fah
  • liver fibrosis e.g., in the absence of nitisinone or any
  • the non-human animals described herein can comprise an accumulation of toxic metabolites such as fumarylacetoacetate and maleylacetoacetate (e.g., in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency and/or in the absence of transplantation of hepatocytes with a functional copy of Fah).
  • This accumulation can lead to hepatocyte loss, liver failure, and death (e.g., in the absence of nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency and/or in the absence of transplantation of hepatocytes with a functional copy of Fah).
  • Some non-human animals described herein can further comprise nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency.
  • Some non-human animals described herein can also comprise transplanted (i.e., xenotransplanted) hepatocytes or transplanted and expanded hepatocytes (e.g., hepatocytes from a different species than the non-human animal, such as human hepatocytes).
  • the transplanted hepatocytes can repopulate the liver of the non-human animals disclosed herein and can restore liver function (e.g., that was lost or decreased due to the inactivated Fah gene).
  • the non-human animals described herein can comprise xenotransplanted human hepatocytes.
  • the transplanted hepatocytes can be wild type hepatocytes, or they can comprise one or more mutations.
  • the transplanted hepatocytes have a wild type FAH gene or an FAH gene that produces a functional FAH protein.
  • the genetically modified non-human animals comprising xenotransplanted hepatocytes (e.g., human hepatocytes) disclosed herein can comprise modifications to the genetically modified non-human animals and/or the xenotransplanted hepatocytes to restore IL-6/IL-6R signaling pathway or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the genetically modified non-human animal does not comprise (and/or is not modified to comprise) Kupffer cells that are species-matched to the transplanted hepatocytes.
  • the genetically modified non-human animal does not comprise (and/or is not modified to comprise) Kupffer cells in the liver that are species-matched to the transplanted hepatocytes. In some cases, the genetically modified non-human animal does not comprise (and/or is not modified to comprise) a reconstituted immune system that is species-matched to the transplanted hepatocytes. In some cases, the genetically modified non-human animal does not comprise (and/or is not modified to comprise) human Kupffer cells. In some cases, the genetically modified non-human animal does not comprise (and/or is not modified to comprise) human Kupffer cells in the liver.
  • the genetically modified non-human animal does not comprise (and/or is not modified to comprise) a reconstituted human immune system. In some cases, the genetically modified non-human animal does not comprise (and/or is not modified to comprise) Kupffer cells that are species-compatible to the transplanted hepatocytes (i.e., that produce IL-6 compatible with the IL-6R in the transplanted hepatocytes). In some cases, the genetically modified non-human animal does not comprise (and/or is not modified to comprise) Kupffer cells in the liver that are species-compatible to the transplanted hepatocytes. In some cases, the genetically modified non-human animal does not comprise (and/or is not modified to comprise) a reconstituted immune system that is species-compatible to the transplanted hepatocytes.
  • the genetically modified non-human animals can comprise xenotransplanted hepatocytes (e.g., xenotransplanted and expanded hepatocytes).
  • the xenotransplanted hepatocytes can be from any species other than that of the recipient non-human animal.
  • the xenotransplanted hepatocytes can be human hepatocytes.
  • the xenotransplanted hepatocytes can be non-human primate (NHP) hepatocytes, such as cynomolgus hepatocytes.
  • NHS non-human primate
  • the transplanted hepatocytes can be from a species whose IL-6R is incompatible with the endogenous non-human animal IL-6 (e.g., the transplanted hepatocytes can be human hepatocytes, and the non-human animal can be a mouse or a rat).
  • the cross-species incompatibility between the transplanted hepatocytes and the recipient non-human animals i.e., the suboptimal interaction or lack of reactivity between the recipient non-human animal IL-6 ligands and the IL-6R on the xenotransplanted hepatocytes
  • a steatosis-like phenotype and lipid droplet accumulation i.e., the suboptimal interaction or lack of reactivity between the recipient non-human animal IL-6 ligands and the IL-6R on the xenotransplanted hepatocytes
  • the modifications described herein can result in reduced lipid droplet accumulation and reduced steatosis in the xenotransplanted hepatocytes compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes do not have modifications to restore IL-6/IL-6R signaling pathway or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the modifications can, for example, restore species-matched hepatic IL-6/IL-6R pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes, or can simply restore IL-6/IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the modifications can, for example, restore species-compatible hepatic IL-6/IL-6R pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the xenotransplanted hepatocytes are modified to restore IL-6/IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the transplanted hepatocytes can be modified to express (i.e., ectopically express) non-human animal IL-6R (i.e., IL-6R from the same species as the recipient non-human animal—in other words, IL-6R species-matched to the recipient non-human animal).
  • the transplanted hepatocytes can be modified to comprise a vector comprising an expression construct for the non-human animal IL-6R comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter.
  • a vector comprising an expression construct for the non-human animal IL-6R comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • a lentiviral vector is used.
  • the transplanted hepatocytes can be genetically modified to comprise in their genome a non-human animal IL-6R expression construct comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter.
  • any suitable promoter can be used.
  • a liver-specific promoter or a promoter active in liver cells e.g., hepatocytes
  • promoters include TTR, ALB, and HBV promoters.
  • a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • hCMV human cytomegalovirus
  • CAG chicken beta-actin/CMV enhancer
  • EF1alpha elongation factor-1 alpha
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • Mouse interleukin-6 receptor subunit alpha (also known as IL-6 receptor subunit alpha, IL-6R subunit alpha, IL-6R-alpha, IL-6RA, IL-6R, IL-6R 1, and CD126) is encoded by the gene Il6ra (also known as Il6r).
  • Il6ra also known as Il6r
  • IL-6R is part of the receptor for interleukin 6. It binds to IL-6 with low affinity but does not transduce a signal. Signal activation necessitate an association with IL6ST. Activation leads to the regulation of the immune response, acute-phase reactions and hematopoiesis.
  • Mouse Il6ra maps to 3 F1; 3 39.19 cM on chromosome 3 (NCBI RefSeq Gene ID 16194; Assembly GRCm39 (GCF_000001635.27); location NC_000069.7 (89776631 . . . 89820503, complement).
  • Reference to the mouse Il6ra gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical mouse IL-6R protein has been assigned UniProt accession number P22272 and NCBI Accession No. NP_034689.2 (SEQ ID NO: 41). Reference to mouse IL-6R proteins includes canonical forms as well as all allelic forms and isoforms.
  • An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_010559.3.
  • the coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS38496.1 (SEQ ID NO: 40).
  • Reference to the mouse Il6ra mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • the transplanted hepatocytes can be modified to express (i.e., ectopically express) non-human animal OSMR (i.e., OSMR from the same species as the recipient non-human animal—in other words, OSMR species-matched to the recipient non-human animal).
  • OSMR non-human animal
  • the transplanted hepatocytes can be modified to express (i.e., ectopically express) species-compatible OSMR (i.e., OSMR from a species that is compatible with the OSM produced by the recipient non-human animal, such that the recipient non-human animal OSM can bind to and activate OSMR signaling).
  • the transplanted hepatocytes can be genetically modified to express mouse OSMR or rat OSMR, respectively.
  • the transplanted hepatocytes can be modified to comprise a vector comprising an expression construct for the non-human animal OSMR comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter.
  • Any suitable vector can be used.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • a lentiviral vector is used.
  • the transplanted hepatocytes can be genetically modified to comprise in their genome a non-human animal OSMR expression construct comprising a nucleic acid encoding the non-human animal OSMR operably linked to a promoter.
  • a promoter can be used in the case of genomic modification or in the case of a vector.
  • a liver-specific promoter or a promoter active in liver cells can be used.
  • promoters include TTR, ALB, and HBV promoters.
  • a constitutive promoter can be used.
  • promoters examples include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • hCMV human cytomegalovirus
  • CAG chicken beta-actin/CMV enhancer
  • EF1alpha elongation factor-1 alpha
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • Mouse oncostatin-M-specific receptor subunit beta (also known as oncostatin M receptor or OSMR or OSMRB) is encoded by the gene Osmr (also known as Osmrb or oncostatin M receptor). It is capable of transducing OSM-specific signaling events through association with GP130 and activating STAT3 downstream.
  • Mouse Osmr maps to 15 A1; 15 3.3 cM on chromosome 15 (NCBI RefSeq Gene ID 18414; Assembly GRCm39 (GCF_000001635.27); location NC_000081.7 (6843049 . . . 6904434, complement).
  • mouse Osmr gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical mouse OSMR protein has been assigned UniProt accession number 070458 and NCBI Accession No. NP_035149.2 (SEQ ID NO: 53).
  • Reference to mouse OSMR proteins includes canonical forms as well as all allelic forms and isoforms.
  • An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_011019.4.
  • the coding sequence for the canonical isoform is assigned CCDS Accession No. CCDS27368.1 (SEQ ID NO: 52).
  • Reference to the mouse Osmr mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • Rat Osmr maps to 2q16 on chromosome 2 (NCBI RefSeq Gene ID 310132; Assembly mRatBN7.2 (GCF_015227675.2); location NC_051337.1 (55907119 . . . 55961373, complement).
  • Reference to the rat Osmr gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • Rat OSMR protein has been assigned UniProt accession number Q65Z14.
  • the canonical rat OSMR protein has been assigned NCBI Accession No. NP_001005384.1 (SEQ ID NO: 55).
  • Reference to rat OSMR proteins includes canonical forms as well as all allelic forms and isoforms.
  • mRNA encoding the canonical isoform
  • NCBI Accession No. NM_001005384.1 NCBI Accession No. NM_001005384.1.
  • a coding sequence for the canonical isoform is set forth in SEQ ID NO: 54.
  • Reference to the rat Osmr mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • the transplanted hepatocytes can be modified to express (i.e., ectopically express) species-compatible IL-6R and OSMR (i.e., IL-6R and OSMR from a species that is compatible with the IL-6 and OSM, respectively, produced by the recipient non-human animal, such that the recipient non-human animal IL-6 and OSM can bind to and activate IL-6R and OSMR signaling, respectively).
  • the transplanted hepatocytes can be modified to express (i.e., ectopically express) the non-human animal receptors for IL-6, LIF, OSM, CNTF, IL-11, CTF1, BSF3, or any combination thereof.
  • the transplanted hepatocytes can be modified to express (i.e., ectopically express) non-human animal Growth Hormone Receptor (GHR) (i.e., GHR from the same species as the recipient non-human animal—in other words, GHR species-matched to the recipient non-human animal).
  • GHR Growth Hormone Receptor
  • the transplanted hepatocytes can be modified to express (i.e., ectopically express) species-compatible GHR (i.e., GHR from a species that is compatible with the GH produced by the recipient non-human animal, such that the recipient non-human animal GH can bind to and activate GHR signaling).
  • the transplanted hepatocytes can be genetically modified to express mouse GHR or rat GHR, respectively.
  • the transplanted hepatocytes can be modified to comprise a vector comprising an expression construct for the non-human animal GHR comprising a nucleic acid encoding the non-human animal GHR operably linked to a promoter.
  • Any suitable vector can be used.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • a lentiviral vector is used.
  • the transplanted hepatocytes can be genetically modified to comprise in their genome a non-human animal GHR expression construct comprising a nucleic acid encoding the non-human animal GHR operably linked to a promoter.
  • a promoter can be used in the case of genomic modification or in the case of a vector.
  • a liver-specific promoter or a promoter active in liver cells can be used.
  • promoters include TTR, ALB, and HBV promoters.
  • a constitutive promoter can be used.
  • promoters examples include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • hCMV human cytomegalovirus
  • CAG chicken beta-actin/CMV enhancer
  • EF1alpha elongation factor-1 alpha
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • transplanted hepatocytes can be modified to express (i.e., ectopically express) a ligand-independent, constitutively active form of IL-6R co-receptor glycoprotein 130 (GP130).
  • GP130 IL-6R co-receptor glycoprotein 130
  • the transplanted hepatocytes are human hepatocytes
  • the constitutively active form of GP130 can be a human GP130.
  • the transplanted hepatocytes can be modified to comprise a vector comprising an expression construct for the constitutively active form of GP130 comprising a nucleic acid encoding the constitutively active form of GP130 operably linked to a promoter. Any suitable vector can be used.
  • Human interleukin-6 receptor subunit beta (also known as IL6ST, IL-6 receptor subunit beta, IL-6R subunit beta, IL-6R-beta, IL-6RB, CDw130, interleukin-6 signal transducer, membrane glycoprotein 130 (gp130), CD130, and oncostatin-M receptor subunit alpha) is encoded by the gene IL6ST.
  • the receptor systems for IL-6, LIF, OSM, CNTF, IL-11, CTF1, and BSF3 can utilize IL6ST (GP130) for initiating signal transmission.
  • IL6ST GP130 binding of IL-6 to IL-6R induces IL6ST (GP130) homodimerization and formation of a high-affinity receptor complex, which activates the intracellular JAK-MAPK and JAK-STAT3 signaling pathways.
  • Human IL6ST maps to 5q11.2 on chromosome 5 (NCBI RefSeq Gene ID 3572; Assembly GRCh38.p14 (GCF_000001405.40); location NC_000005.10 (55935095 . . . 55994963, complement).
  • Reference to the human IL6ST gene includes the canonical, wild type form as well as all allelic forms and isoforms.
  • the canonical human IL6ST (GP130) protein has been assigned UniProt accession number P40189 and NCBI Accession No. NP_002175.2 (SEQ ID NO: 49).
  • Reference to human IL6ST (GP130) proteins includes canonical forms as well as all allelic forms and isoforms.
  • An mRNA (cDNA) encoding the canonical isoform is assigned NCBI Accession No. NM_002184.3.
  • a coding sequence encoding the canonical isoform is assigned CCDS Accession No. CCDS3971.1 (SEQ ID NO: 48).
  • Reference to the human IL6ST mRNA (cDNA) and coding sequence includes the canonical forms as well as all allelic forms and isoforms.
  • the constitutively active human GP130 comprises a deletion of the region of GP130 from Tyr186 to Tyr190 (GP130 Y186-Y190del ).
  • An exemplary GP130 Y186-Y190del protein is set forth in SEQ ID NO: 47 and is encoded by the coding sequence set forth in SEQ ID NO: 46.
  • the recipient non-human animal is modified to restore IL-6/IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the non-human animal can comprise IL-6 from the species of the xenotransplanted hepatocytes (e.g., IL-6 that is species-matched with the xenotransplanted hepatocytes).
  • the non-human animal can comprise IL-6 compatible with the species of the xenotransplanted hepatocytes (e.g., IL-6 that is species-compatible with the xenotransplanted hepatocytes, such that the IL-6 can activate IL-6R signaling in the xenotransplanted hepatocytes).
  • the non-human animal can comprise human IL-6 (or a human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6), such as cynomolgus IL-6) if the xenotransplanted hepatocytes are human hepatocytes.
  • the non-human animal can comprise human IL-6 (or a human-IL-6R-compatible IL-6, such as cynomolgus IL-6) if the xenotransplanted hepatocytes are human hepatocytes.
  • Human IL-6 is described above.
  • the IL-6 can be, for example, in the serum of the non-human animal or the liver of the non-human animal.
  • the IL-6 can be expressed by any suitable cell type in the non-human animal. In one example, the IL-6 is expressed in muscle cells in the non-human animal.
  • the non-human animal comprises a vector comprising an expression construct for species-matched IL-6 (e.g., human IL-6) comprising a nucleic acid encoding the species-matched IL-6 (e.g., human IL-6) operably linked to a promoter.
  • the non-human animal can comprise a vector comprising an expression construct for species-compatible IL-6 (e.g., the IL-6 is compatible with the IL-6R expressed by the transplanted hepatocytes such that the IL-6 can activate IL-6R signaling in the hepatocytes) comprising a nucleic acid encoding the species-compatible IL-6 operably linked to a promoter.
  • the non-human animal can comprise a vector comprising an expression construct for human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) (e.g., cynomolgus IL-6) comprising a nucleic acid encoding the human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) operably linked to a promoter.
  • human-IL-6R-compatible ligand e.g., human-IL-6R-compatible IL-6
  • a vector comprising an expression construct for human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) (e.g., cynomolgus IL-6) comprising a nucleic acid encoding the human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) operably linked to a promoter.
  • human-IL-6R-compatible ligand
  • the non-human animal can comprise a vector comprising an expression construct for human-IL-6R-compatible IL-6 (e.g., cynomolgus IL-6) comprising a nucleic acid encoding the human-IL-6R-compatible IL-6 operably linked to a promoter.
  • the non-human animal can comprise the vector in muscle cells.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV vector is used, such as an AAV serotype for expression in muscle.
  • a recombinant AAV9 vector is used.
  • the non-human animal can comprise in its genome a species-matched IL-6 (e.g., human IL-6) expression construct comprising a nucleic acid encoding the species-matched IL-6 (e.g., human IL-6) operably linked to a promoter.
  • the non-human animal can comprise in its genome a species-compatible IL-6 (e.g., the IL-6 is compatible with the IL-6R expressed by the transplanted hepatocytes such that the IL-6 can activate IL-6R signaling in the hepatocytes) expression construct comprising a nucleic acid encoding the species-compatible IL-6 operably linked to a promoter.
  • a species-compatible IL-6 e.g., the IL-6 is compatible with the IL-6R expressed by the transplanted hepatocytes such that the IL-6 can activate IL-6R signaling in the hepatocytes
  • expression construct comprising a nucleic acid encoding the species-compatible IL-6 operably linked to a promoter.
  • the non-human animal can comprise in its genome a human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) (e.g., cynomolgus IL-6) expression construct comprising a nucleic acid encoding the human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) operably linked to a promoter.
  • a human-IL-6R-compatible ligand e.g., human-IL-6R-compatible IL-6
  • cynomolgus IL-6 expression construct comprising a nucleic acid encoding the human-IL-6R-compatible ligand (e.g., human-IL-6R-compatible IL-6) operably linked to a promoter.
  • the non-human animal can comprise in its genome a human-IL-6R-compatible IL-6 (e.g., cynomolgus IL-6) expression construct comprising a nucleic acid encoding the human-IL-6R-compatible IL-6 operably linked to a promoter.
  • a human-IL-6R-compatible IL-6 e.g., cynomolgus IL-6
  • the expression construct could be at a safe harbor locus in the non-human animal.
  • any suitable promoter can be used.
  • a tissue-specific promoter can be used.
  • a muscle-specific promoter or a promoter active in muscle cells can be used.
  • a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein.
  • a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • hCMV human cytomegalovirus
  • CAG chicken beta-actin/CMV enhancer
  • EF1alpha elongation factor-1 alpha
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • the non-human animal can comprise OSM from the species of the xenotransplanted hepatocytes (e.g., OSM that is species-matched with the xenotransplanted hepatocytes).
  • the non-human animal can comprise OSM compatible with the species of the xenotransplanted hepatocytes (e.g., OSM that is species-compatible with the xenotransplanted hepatocytes, such that the OSM can activate OSMR signaling in the xenotransplanted hepatocytes).
  • the non-human animal can comprise human OSM (or a human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM)) if the xenotransplanted hepatocytes are human hepatocytes.
  • Human OSM is described above.
  • the OSM can be, for example, in the serum of the non-human animal or the liver of the non-human animal.
  • the OSM can be expressed by any suitable cell type in the non-human animal. In one example, the OSM is expressed in muscle cells in the non-human animal.
  • the non-human animal can comprise a vector comprising an expression construct for human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) (e.g., cynomolgus OSM) comprising a nucleic acid encoding the human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) operably linked to a promoter.
  • human-OSMR-compatible OSM e.g., human-OSMR-compatible OSM
  • the non-human animal can comprise the vector in muscle cells.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • an AAV vector is used, such as an AAV serotype for expression in muscle.
  • a recombinant AAV9 vector is used.
  • the non-human animal can comprise in its genome a species-matched OSM (e.g., human OSM) expression construct comprising a nucleic acid encoding the species-matched OSM (e.g., human OSM) operably linked to a promoter.
  • a species-matched OSM e.g., human OSM
  • a nucleic acid encoding the species-matched OSM (e.g., human OSM) operably linked to a promoter.
  • the non-human animal can comprise in its genome a species-compatible OSM (e.g., the OSM is compatible with the OSMR expressed by the transplanted hepatocytes such that the OSM can activate OSMR signaling in the hepatocytes) expression construct comprising a nucleic acid encoding the species-compatible OSM operably linked to a promoter.
  • a species-compatible OSM e.g., the OSM is compatible with the OSMR expressed by the transplanted hepatocytes such that the OSM can activate OSMR signaling in the hepatocytes
  • expression construct comprising a nucleic acid encoding the species-compatible OSM operably linked to a promoter.
  • the non-human animal can comprise in its genome a human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) (e.g., cynomolgus OSM) expression construct comprising a nucleic acid encoding the human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) operably linked to a promoter.
  • a human-OSMR-compatible ligand e.g., human-OSMR-compatible OSM
  • cynomolgus OSM cynomolgus OSM
  • the expression construct could be at a safe harbor locus in the non-human animal.
  • any suitable promoter can be used.
  • a tissue-specific promoter can be used.
  • a muscle-specific promoter or a promoter active in muscle cells can be used.
  • An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein.
  • a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • the recipient non-human animal is modified to restore IL-6/IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the non-human animal can comprise a GP130 activator (e.g., human GP130 activator), such as a GP130-activating ligand (e.g., human-GP130-activating ligand).
  • the non-human animal can comprise one or more GP130-activating ligands.
  • the non-human animal can comprise two or more GP130-activating ligands.
  • the non-human animal can comprise three or more GP130-activating ligands.
  • the non-human animal can comprise four or more GP130-activating ligands.
  • Ligands that activate GP130 are known.
  • IL-6, LIF, OSM, CNTF, IL-11, CTF1, and BSF3 are all ligands that can activate GP130.
  • the non-human animals can comprise a ligand (e.g., IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), interleukin-11 (IL-11), cardiotrophin-1 (CTF1), or cardiotrophin-like cytokine factor 1 (BSF3)) from the species of the xenotransplanted hepatocytes (e.g., IL-6, LIF, OSM, CNTF, IL-11, CTF1, or BSF3 that is species-matched or species-compatible with the xenotransplanted hepatocytes).
  • a ligand e.g., IL-6, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), interleukin-11 (IL-11), cardiotrophin-1 (CTF1), or cardiotrophin-like cytokine factor 1 (BSF3)
  • a ligand e.g.,
  • the non-human animals can comprise IL-6 (e.g., species-matched or species-compatible with the xenotransplanted hepatocytes).
  • the non-human animals can comprise OSM (e.g., species-matched or species-compatible with the xenotransplanted hepatocytes).
  • the non-human animals can comprise IL-6 and OSM (e.g., species-matched or species-compatible with the xenotransplanted hepatocytes).
  • the non-human animals can comprise IL-6, LIF, OSM, CNTF, IL-11, CTF1, BSF3, or any combination thereof (e.g., species-matched or species-compatible with the xenotransplanted hepatocytes).
  • the non-human animal can comprise human IL-6, LIF, OSM, CNTF, IL-11, CTF1, or BSF3 if the xenotransplanted hepatocytes are human hepatocytes.
  • a GP130 activator is a GP130 agonist antibody or antigen-binding protein (e.g., human GP130 agonist antibody or antigen-binding protein). GP130-activating antibodies are known.
  • GP130 activator is a chimeric GP130 ligand, termed IC7Fc, where one GP130 binding site has been removed from IL-6 and replaced with the leukemia inhibitory factor receptor (LIFR) binding site from CNTF and then fused with the fragment crystallizable (Fc) domain of immunoglobulin G (IgG).
  • LIFR leukemia inhibitory factor receptor
  • Fc fragment crystallizable domain of immunoglobulin G
  • the GP130 activator (e.g., ligand) can be, for example, in the serum of the non-human animal or the liver of the non-human animal.
  • the GP130 activator (e.g., ligand) can be expressed by any suitable cell type in the non-human animal.
  • the GP130 activator (e.g., ligand) is expressed in muscle cells in the non-human animal.
  • the non-human animal comprises a vector comprising an expression construct for the GP130 activator (e.g., ligand) comprising a nucleic acid encoding the GP130 activator operably linked to a promoter.
  • the non-human animal can comprise the vector in muscle cells. Any suitable vector can be used.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV vector is used, such as an AAV serotype for expression in muscle.
  • a recombinant AAV9 vector is used.
  • the non-human animal can comprise in its genome a GP130 activator (e.g. ligand) expression construct comprising a nucleic acid encoding the GP130 activator (e.g. ligand) operably linked to a promoter.
  • the expression construct could be at a safe harbor locus in the non-human animal.
  • any suitable promoter can be used.
  • a tissue-specific promoter can be used.
  • a muscle-specific promoter or a promoter active in muscle cells can be used.
  • An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein.
  • a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • the non-human animal can comprise Growth Hormone (GH) from the species of the xenotransplanted hepatocytes (e.g., GH that is species-matched with the xenotransplanted hepatocytes).
  • GH Growth Hormone
  • the non-human animal can comprise GH compatible with the species of the xenotransplanted hepatocytes (e.g., GH that is species-compatible with the xenotransplanted hepatocytes, such that the GH can activate GHR signaling in the xenotransplanted hepatocytes).
  • the non-human animal can comprise human GH (or a human-GHR-compatible GH) if the xenotransplanted hepatocytes are human hepatocytes.
  • the GH can be, for example, in the serum of the non-human animal or the liver of the non-human animal.
  • the GH can be expressed by any suitable cell type in the non-human animal.
  • the GH is expressed in muscle cells in the non-human animal.
  • the non-human animal comprises a vector comprising an expression construct for species-matched GH (e.g., human GH) comprising a nucleic acid encoding the species-matched GH (e.g., human GH) operably linked to a promoter.
  • the non-human animal can comprise a vector comprising an expression construct for species-compatible GH (e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes such that the GH can activate GHR signaling in the hepatocytes) comprising a nucleic acid encoding the species-compatible GH operably linked to a promoter.
  • species-compatible GH e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes such that the GH can activate GHR signaling in the hepatocytes
  • the non-human animal can comprise a vector comprising an expression construct for human-GHR-compatible GH (e.g., cynomolgus GH) comprising a nucleic acid encoding the human-GHR-compatible GH operably linked to a promoter.
  • the non-human animal can comprise the vector in muscle cells.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • an AAV vector is used, such as an AAV serotype for expression in muscle.
  • a recombinant AAV9 vector is used.
  • the non-human animal can comprise in its genome a species-matched GH (e.g., human GH) expression construct comprising a nucleic acid encoding the species-matched GH (e.g., human GH) operably linked to a promoter.
  • the non-human animal can comprise in its genome a species-compatible GH (e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes such that the GH can activate GHR signaling in the hepatocytes) expression construct comprising a nucleic acid encoding the species-compatible GH operably linked to a promoter.
  • a species-compatible GH e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes such that the GH can activate GHR signaling in the hepatocytes
  • the non-human animal can comprise in its genome a human-GHR-compatible GH (e.g., cynomolgus GH) expression construct comprising a nucleic acid encoding the human-GHR-compatible GH operably linked to a promoter.
  • the expression construct could be at a safe harbor locus in the non-human animal.
  • any suitable promoter can be used.
  • a tissue-specific promoter can be used.
  • a muscle-specific promoter or a promoter active in muscle cells can be used.
  • An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein.
  • MHCK muscle creatine kinase
  • a constitutive promoter can be used.
  • the non-human animal can comprise a humanized IL6 locus as described elsewhere herein.
  • Examples of non-human animals with humanized IL6 genes are provided, e.g., in U.S. Pat. No. 9,622,460, herein incorporated by reference in its entirety for all purposes.
  • the non-human animal can be, in some cases, homozygous for the humanized IL6 gene. In other cases, the non-human animal can be heterozygous for the humanized IL6 gene.
  • the non-human animal can comprise the humanized IL6 gene in its germline.
  • the humanized IL6 gene can comprise a human IL6 nucleic acid encoding a human IL-6 protein (e.g., a fully human IL-6 protein).
  • the human OSM nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human OSM gene from the start codon to the stop codon, or can comprise a human OSM complementary DNA (cDNA).
  • the human OSM nucleic acid can be inserted into the non-human animal Osm genomic locus, or it can replace a corresponding region of the non-human animal Osm locus (e.g., a region of a human OSM gene from the start codon to the stop codon can replace a region of the non-human animal Osm gene from the start codon to the stop codon).
  • the non-human animal can comprise a humanized IL6 locus as described elsewhere herein and a humanized OSM locus as described elsewhere herein.
  • the non-human animal can comprise a humanized IL6 locus as described elsewhere herein and a humanized GH locus as described elsewhere herein.
  • the non-human animal can comprise a humanized OSM locus as described elsewhere herein and a humanized GH locus as described elsewhere herein.
  • the modifications described herein can result in reduced lipid droplet accumulation and reduced steatosis in the xenotransplanted hepatocytes compared to non-human animals in which the genetically modified non-human animals and the xenotransplanted hepatocytes do not have modifications to restore IL-6/IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the modifications can, for example, restore species-matched hepatic IL-6/IL-6R pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes, or can simply restore IL-6/IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the modifications can, for example, restore species-compatible hepatic IL-6/IL-6R pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the transplanted hepatocytes/hepatocyte progenitors can be genetically modified to express mouse IL-6R or rat IL-6R, respectively.
  • the transplanted hepatocytes/hepatocyte progenitors can be modified to comprise a vector comprising an expression construct for the non-human animal IL-6R comprising a nucleic acid encoding the non-human animal IL-6R operably linked to a promoter.
  • Any suitable vector can be used.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • promoters examples include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • hCMV human cytomegalovirus
  • CAG chicken beta-actin/CMV enhancer
  • EF1alpha elongation factor-1 alpha
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • the transplanted hepatocytes/hepatocyte progenitors can be genetically modified to comprise in their genome a GP130 expression construct comprising a nucleic acid encoding the constitutively active form of GP130 operably linked to a promoter.
  • the expression construct could be at a safe harbor locus in the non-human animal.
  • any suitable promoter can be used.
  • a liver-specific promoter or a promoter active in liver cells e.g., hepatocytes
  • examples of such promoters include TTR, ALB, and HBV promoters.
  • a constitutive promoter can be used.
  • the non-human animal can comprise a vector comprising an expression construct for human-IL-6R-compatible IL-6 (e.g., cynomolgus IL-6) comprising a nucleic acid encoding the human-IL-6R-compatible IL-6 operably linked to a promoter.
  • the non-human animal can comprise the vector in muscle cells.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV vector is used, such as an AAV serotype for expression in muscle.
  • the non-human animal can comprise OSM species-compatible with the species of the xenotransplanted hepatocytes/hepatocyte progenitors (e.g., OSM that can activate OSMR signaling in the xenotransplanted hepatocytes/hepatocyte progenitors).
  • OSM xenotransplanted hepatocytes/hepatocyte progenitors
  • the non-human animal can comprise human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) (e.g., cynomolgus OSM) if the xenotransplanted hepatocytes/hepatocyte progenitors are human hepatocytes/hepatocyte progenitors.
  • Human OSM is described above.
  • a muscle-specific promoter or a promoter active in muscle cells can be used.
  • An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MI-1) and muscle creatine kinase (CK) promoter (MHCK7) as described herein.
  • a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • the recipient non-human animal is modified to restore IL-6/IL-6R signaling pathway activity or GP130 signaling pathway activity in the xenotransplanted hepatocytes.
  • the non-human animal can comprise a GP130 activator (e.g., human GP130 activator), such as a GP130-activating ligand (e.g., human-GP130-activating ligand).
  • the non-human animal can comprise one or more GP130-activating ligands.
  • the non-human animal can comprise two or more GP130-activating ligands.
  • the non-human animal can comprise three or more GP130-activating ligands.
  • the non-human animal can comprise four or more GP130-activating ligands.
  • Ligands that activate GP130 are known.
  • IL-6, LIF, OSM, CNTF, IL-11, CTF1, and BSF3 are all ligands that can activate GP130.
  • GP130 activator is a chimeric GP130 ligand, termed IC7Fc, where one GP130 binding site has been removed from IL-6 and replaced with the leukemia inhibitory factor receptor (LIFR) binding site from CNTF and then fused with the fragment crystallizable (Fc) domain of immunoglobulin G (IgG).
  • LIFR leukemia inhibitory factor receptor
  • Fc fragment crystallizable domain of immunoglobulin G
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV vector is used, such as an AAV serotype for expression in muscle.
  • a recombinant AAV9 vector is used.
  • the non-human animal can comprise in its genome a GP130 activator (e.g. ligand) expression construct comprising a nucleic acid encoding the GP130 activator (e.g. ligand) operably linked to a promoter.
  • the expression construct could be at a safe harbor locus in the non-human animal.
  • the non-human animal can comprise GH species-compatible with the species of the xenotransplanted hepatocytes/hepatocyte progenitors (e.g., GH that can activate GHR signaling in the xenotransplanted hepatocytes/hepatocyte progenitors).
  • the non-human animal can comprise human-GHR-compatible GH (e.g., cynomolgus GH) if the xenotransplanted hepatocytes/hepatocyte progenitors are human hepatocytes/hepatocyte progenitors.
  • the GH can be, for example, in the serum of the non-human animal or the liver of the non-human animal.
  • the GH can be expressed by any suitable cell type in the non-human animal.
  • the GH is expressed in muscle cells in the non-human animal.
  • the non-human animal comprises a vector comprising an expression construct for species-matched GH (e.g., human GH) comprising a nucleic acid encoding the species-matched GH (e.g., human GH) operably linked to a promoter.
  • the non-human animal can comprise a vector comprising an expression construct for species-compatible GH (e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes such that the GH can activate GHR signaling in the hepatocytes) comprising a nucleic acid encoding the species-compatible GH operably linked to a promoter.
  • species-compatible GH e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes such that the GH can activate GHR signaling in the hepatocytes
  • the non-human animal can comprise a vector comprising an expression construct for human-GHR-compatible GH (e.g., cynomolgus GH) comprising a nucleic acid encoding the human-GHR-compatible GH operably linked to a promoter.
  • the non-human animal can comprise the vector in muscle cells.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • an AAV vector is used, such as an AAV serotype for expression in muscle.
  • a recombinant AAV9 vector is used.
  • the non-human animal can comprise in its genome a species-matched GH (e.g., human GH) expression construct comprising a nucleic acid encoding the species-matched GH (e.g., human GH) operably linked to a promoter.
  • the non-human animal can comprise in its genome a species-compatible GH (e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes such that the GH can activate GHR signaling in the hepatocytes) expression construct comprising a nucleic acid encoding the species-compatible GH operably linked to a promoter.
  • a species-compatible GH e.g., the GH is compatible with the GHR expressed by the transplanted hepatocytes such that the GH can activate GHR signaling in the hepatocytes
  • the non-human animal can comprise in its genome a human-GHR-compatible GH (e.g., cynomolgus GH) expression construct comprising a nucleic acid encoding the human-GHR-compatible GH operably linked to a promoter.
  • the expression construct could be at a safe harbor locus in the non-human animal.
  • any suitable promoter can be used.
  • a tissue-specific promoter can be used.
  • a muscle-specific promoter or a promoter active in muscle cells can be used.
  • An example of a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein.
  • MHCK muscle creatine kinase
  • a constitutive promoter can be used.
  • promoters examples include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • hCMV human cytomegalovirus
  • CAG chicken beta-actin/CMV enhancer
  • EF1alpha elongation factor-1 alpha
  • an inducible promoter can be used.
  • an exogenous promoter can be used or an endogenous promoter at the target genomic locus (e.g., a safe harbor locus) can be used.
  • the human IL6 nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human IL6 gene from the start codon to the stop codon, or can comprise a human IL6 complementary DNA (cDNA).
  • the human IL6 nucleic acid can be inserted into the non-human animal 116 genomic locus, or it can replace a corresponding region of the non-human animal 116 locus (e.g., a region of a human IL6 gene from the start codon to the stop codon can replace a region of the non-human animal 116 gene from the start codon to the stop codon).
  • the humanized IL6 gene (or the human IL6 nucleic acid) can be operably linked to the endogenous non-human animal 116 promoter. In other words, expression of the humanized IL6 gene can be driven by the endogenous non-human animal 116 promoter.
  • the non-human animal can comprise a humanized OSM locus as described elsewhere herein.
  • the non-human animal can be, in some cases, homozygous for the humanized OSM gene. In other cases, the non-human animal can be heterozygous for the humanized OSM gene.
  • the non-human animal can comprise the humanized OSM gene in its germline.
  • the humanized OSM gene can comprise a human OSM nucleic acid encoding a human OSM protein (e.g., a fully human OSM protein).
  • the human OSM nucleic acid can be a genomic nucleic acid, such as a genomic nucleic acid comprising a region of a human OSM gene from the start codon to the stop codon, or can comprise a human OSM complementary DNA (cDNA).
  • the human OSM nucleic acid can be inserted into the non-human animal Osm genomic locus, or it can replace a corresponding region of the non-human animal Osm locus (e.g., a region of a human OSM gene from the start codon to the stop codon can replace a region of the non-human animal Osm gene from the start codon to the stop codon).
  • the humanized OSM gene (or the human OSM nucleic acid) can be operably linked to the endogenous non-human animal Osm promoter. In other words, expression of the humanized OSM gene can be driven by the endogenous non-human animal Osm promoter.
  • the non-human animal can comprise a humanized IL6 locus as described elsewhere herein and a humanized OSM locus as described elsewhere herein.
  • the non-human animal can comprise a humanized GH locus. Similar to IL6, mGH shows species specificities in receptor binding, and administration of human Growth Hormone (GH) in humanized liver mice could correct fatty liver in humanized liver mice. Tateno et al. (2011) Endocrinology 152:1479-1491, herein incorporated by reference in its entirety for all purposes.
  • GH Growth Hormone
  • the non-human animal can comprise a humanized IL6 locus as described elsewhere herein and a humanized GH locus as described elsewhere herein.
  • the non-human animal can comprise a humanized OSM locus as described elsewhere herein and a humanized GH locus as described elsewhere herein.
  • Exogenous urokinase plasminogen activator (uPA; also called urokinase) or an exogenous nucleic acid encoding uPA can be administered to the non-human animal prior to transplantation to prime the liver for improved repopulation by human hepatocytes.
  • any other compound that primes the liver for improved repopulation by xenotransplanted hepatocytes e.g., human hepatocytes
  • an adenovirus or an adeno-associated virus (AAV) or adenoviral or AAV vector encoding urokinase plasminogen activator is administered to the non-human animal prior to transplantation.
  • the exogenous uPA or exogenous nucleic acid encoding uPA can be administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days prior to transplantation.
  • the exogenous uPA or exogenous nucleic acid encoding uPA can be administered about 24 hours to about 48 hours prior to transplantation.
  • the uPA can be human uPA, and it can be a secreted form or can be a non-secreted form.
  • the nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency can be administered for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, or at least about 4 weeks after transplantation.
  • nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency can be withdrawn (i.e., no longer administered to the non-human animal) prior to the transplantation.
  • nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency can be withdrawn at least about 12 hours, at least about 24 hours, at least about 1 day, at least about 2 days, or at least about 3 days prior to transplantation, or no more than about 12 hours, no more than about 24 hours, no more than about 1 day, no more than about 2 days, or no more than about 3 days prior to transplantation.
  • nitisinone or any other compound that ameliorates toxicity caused by Fah deficiency can be cycled off and on to promote repopulation by the transplanted hepatocytes.
  • the cycling can be about 3 days to about 9 days, about 4 days to about 8 days, about 5 days to about 7 days, about 3 days to about 7 days, about 4 days to about 7 days, about 5 days to about 8 days, about 5 days to about 9 days, or about 6 days off, and can be about 1 day to about 5 days, about 2 days to about 4 days, about 1 day to about 4 days, about 1 day to about 3 days, about 3 days to about 4 days, about 3 days to about 5 days, or about 3 days on.
  • the hepatocytes or hepatocyte progenitors can be allowed to expand for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.
  • the recipient non-human animals receiving the transplanted hepatocytes or hepatocyte progenitors can be any suitable age.
  • the non-human animals e.g., mice or rats
  • the non-human animals can be at least about 1 day old, at least about 2 days old, at least about 1 week old, at least about 2 weeks old, at least about 3 weeks old, at least about 4 weeks old, at least about 1 month old, or at least about 2 months old.
  • Such methods can comprise, for example, administering IL-6 that is species-matched to the xenotransplanted hepatocytes or a nucleic acid encoding the IL-6 (e.g., administering human IL-6 or a nucleic acid encoding the human IL-6) to the non-human animal, wherein the species-matched IL-6 restores interleukin-6 (IL-6)/interleukin-6 receptor (IL-6R) signaling pathway activity or GP130 signaling pathway activity in the transplanted hepatocytes.
  • IL-6 interleukin-6
  • IL-6R interleukin-6 receptor
  • any suitable administration method can be used, and such methods are disclosed in more detail elsewhere herein.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • an AAV vector is used, such as an AAV serotype for expression in muscle.
  • a recombinant AAV9 vector is used.
  • Any suitable promoter can be used.
  • a tissue-specific promoter can be used.
  • a muscle-specific promoter or a promoter active in muscle cells can be used.
  • such methods can comprise, for example, administering human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) (e.g., cynomolgus OSM) or a nucleic acid encoding the human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) (e.g., administering human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) or a nucleic acid encoding the human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM)) to the non-human animal, wherein the human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) restores GP130 signaling pathway activity in the transplanted hepatocytes.
  • human-OSMR-compatible ligand e.g., human-OSMR-compatible OSM
  • human-OSMR-compatible OSM e.g., human-OSMR-
  • the nucleic acid is administered, wherein the nucleic acid comprises an expression construct (e.g., a vector comprising an expression construct) for species-matched OSM (e.g., human OSM, or human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) if the transplanted hepatocytes are human hepatocytes) comprising a nucleic acid encoding the species-matched OSM (e.g., human OSM, or human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) if the transplanted hepatocytes are human hepatocytes) operably linked to a promoter.
  • species-matched OSM e.g., human OSM, or human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) if the transplanted hepatocytes are human hepatocytes
  • a promoter e.g., a promoter
  • the species-matched OSM protein e.g., human OSM, or human-OSMR-compatible ligand (e.g., human-OSMR-compatible OSM) if the transplanted hepatocytes are human hepatocytes
  • the OSM protein can be administered, e.g., to the liver or such that it reaches the liver of the non-human animal. Suitable methods for administering are described in more detail elsewhere herein.
  • a muscle-specific promoter is a hybrid mouse alpha-myosin heavy-chain (MH) and muscle creatine kinase (CK) promoter (MHCK7) as described herein.
  • MHCK7 muscle creatine kinase promoter
  • a liver-specific promoter or a promoter active in liver cells e.g., hepatocytes
  • promoters are described elsewhere herein.
  • a constitutive promoter can be used. Examples of such promoters include human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), and elongation factor-1 alpha (EF1alpha).
  • an inducible promoter can be used.
  • the species-matched GH protein e.g., human GH, or human-GHR-compatible GH if the transplanted hepatocytes are human hepatocytes
  • the GH protein can be administered, e.g., to the liver or such that it reaches the liver of the non-human animal. Suitable methods for administering are described in more detail elsewhere herein.
  • GP130 activator is a chimeric GP130 ligand, termed IC7Fc, where one GP130 binding site has been removed from IL-6 and replaced with the leukemia inhibitory factor receptor (LIFR) binding site from CNTF and then fused with the fragment crystallizable (Fc) domain of immunoglobulin G (IgG).
  • LIFR leukemia inhibitory factor receptor
  • Fc fragment crystallizable domain of immunoglobulin G
  • the GP130 activator can be expressed by any suitable cell type in the non-human animal.
  • the GP130 activator e.g., ligand
  • the GP130 activator is expressed in muscle cells in the non-human animal.
  • a vector comprising an expression construct for the GP130 activator (e.g., ligand) comprising a nucleic acid encoding the GP130 activator operably linked to a promoter is administered.
  • the vector is administered to muscle cells.
  • Any suitable vector can be used.
  • the vector can be a viral vector, such as a lentiviral vector or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • Various methods are provided for assessing activity of human-liver-targeting agents/reagents in vivo using the genetically modified non-human animals comprising human hepatocytes (i.e., xenotransplanted human hepatocytes) or a humanized liver as described elsewhere herein.
  • Such methods can comprise: (a) administering the human-liver-targeting reagent to the non-human animal; and (b) assessing the activity of the human-liver-targeting reagent in the liver of the non-human animal.
  • a human-liver-targeting reagent can be any reagent that targets a human liver, a cell in a human liver, a protein expressed in a human liver, an RNA expressed in a human liver, a gene expressed in a human liver, or any other target present in a human liver.
  • a non-limiting example of a target gene expressed in the liver is albumin.
  • a human-liver-targeting reagent can be an antigen-binding protein (e.g., agonist antibody) targeting a protein or antigen expressed by or present in a human liver.
  • antigen-binding protein e.g., agonist antibody
  • antigen-binding protein includes any protein that binds to an antigen.
  • antigen-binding proteins include an antibody, an antigen-binding fragment of an antibody, a multispecific antibody (e.g., a bi-specific antibody), an scFv, a bis-scFv, a diabody, a triabody, a tetrabody, a V-NAR, a VHH, a VL, a F(ab), a F(ab) 2 , a DVD (dual variable domain antigen-binding protein), an SVD (single variable domain antigen-binding protein), a bispecific T-cell engager (BiTE), or a Davisbody (U.S. Pat. No. 8,586,713, herein incorporated by reference herein in its entirety for all purposes).
  • Other human-liver-targeting reagents include small molecules targeting a cell, protein, RNA, gene, or any other target present in or expressed in a human liver.
  • human-liver-targeting reagents can include genome editing reagents such as a nuclease agent (e.g., a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) nuclease, a zinc finger nuclease (ZFN), or a Transcription Activator-Like Effector Nuclease (TALEN)) that cleaves a recognition site within a target gene expressed in a human liver.
  • a nuclease agent e.g., a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) nuclease, a zinc finger nuclease (ZFN), or a Transcription Activator-Like Effector Nuclease (TALEN)
  • the quantitative assay can be carried out via a quantitative PCR, such as a real-time PCR (qPCR).
  • qPCR real-time PCR
  • the real-time PCR can utilize a first primer set that recognizes the target locus and a second primer set that recognizes a non-targeted reference locus.
  • the primer set can comprise a fluorescent probe that recognizes the amplified sequence.
  • Such methods can also comprise measuring expression levels of the RNA (e.g., messenger RNA) produced by a target genomic locus, or by measuring expression levels of the protein encoded by the target genomic locus.
  • RNA e.g., messenger RNA
  • protein levels can be measured in liver, or if the target genomic locus encodes a secreted protein, secreted levels can be measured in the serum.
  • the human-liver-targeting reagent can target a target gene expressed in human liver, and the method can comprise measuring expression of an RNA (e.g., a messenger RNA) or a protein encoded by the target gene.
  • Agents or compounds introduced into the cell or non-human animal can be provided in compositions comprising a carrier increasing the stability of the introduced molecules (e.g., prolonging the period under given conditions of storage (e.g., ⁇ 20° C., 4° C., or ambient temperature) for which degradation products remain below a threshold, such below 0.5% by weight of the starting nucleic acid or protein; or increasing the stability in vivo).
  • a carrier increasing the stability of the introduced molecules (e.g., prolonging the period under given conditions of storage (e.g., ⁇ 20° C., 4° C., or ambient temperature) for which degradation products remain below a threshold, such below 0.5% by weight of the starting nucleic acid or protein; or increasing the stability in vivo).
  • Non-limiting examples of such carriers include poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates
  • a nucleic acid or protein can be introduced into a non-human animal cell or non-human animal in a carrier such as a poly(lactic acid) (PLA) microsphere, a poly(D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipid cochleate, or a lipid microtubule.
  • a carrier such as a poly(lactic acid) (PLA) microsphere, a poly(D,L-lactic-coglycolic-acid) (PLGA) microsphere, a liposome, a micelle, an inverse micelle, a lipid cochleate, or a lipid microtubule.
  • PLA poly(lactic acid)
  • PLGA poly(D,L-lactic-coglycolic-acid)
  • a liposome e.g., a micelle, an inverse micelle, a lipid cochleate, or
  • lipid nanoparticle LNP-mediated delivery.
  • Lipid formulations can protect biological molecules from degradation while improving their cellular uptake.
  • Lipid nanoparticles are particles comprising a plurality of lipid molecules physically associated with each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension.
  • Administration in vivo can be by any suitable route including, for example, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular.
  • Systemic modes of administration include, for example, oral and parenteral routes.
  • parenteral routes include intravenous, intraarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes.
  • a specific example is intravenous infusion. Nasal instillation and intravitreal injection are other specific examples.
  • Local modes of administration include, for example, intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate or into the putamen), cerebral cortex, precentral gyms, hippocampus (e.g., into the dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum, or substantia nigra), intraocular, intraorbital, subconjuctival, intravitreal, subretinal, and transscleral routes.
  • intraparenchymal e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate or into the putamen)
  • cerebral cortex e.g., into the caudate or into the putamen
  • hippocampus
  • the route of administration is subcutaneous or intravenous.
  • the route of administration is intrasplenic injection.
  • the route of administration is injection into the hepatic artery, the spleen, the portal vein, the peritoneal cavity, hepatic tissue mass, or the lymphatic system, or as part of a liver tissue graft.
  • human-liver-targeting reagents or other agents or compounds e.g., IL-6 or a nucleic acid encoding IL-6, a nucleic acid encoding GP130, a nucleic acid encoding IL-6R, OSM or a nucleic acid encoding OSM, a nucleic acid encoding OSMR, etc.
  • IL-6 or a nucleic acid encoding IL-6 e.g., IL-6 or a nucleic acid encoding IL-6, a nucleic acid encoding GP130, a nucleic acid encoding IL-6R, OSM or a nucleic acid encoding OSM, a nucleic acid encoding OSMR, etc.
  • IL-6 or a nucleic acid encoding IL-6 e.g., IL-6 or a nucleic acid encoding IL-6, a nucleic acid encoding GP130, a nucleic acid en
  • the introduction can be performed at least two times over a period of time, at least three times over a period of time, at least four times over a period of time, at least five times over a period of time, at least six times over a period of time, at least seven times over a period of time, at least eight times over a period of time, at least nine times over a period of times, at least ten times over a period of time, at least eleven times, at least twelve times over a period of time, at least thirteen times over a period of time, at least fourteen times over a period of time, at least fifteen times over a period of time, at least sixteen times over a period of time, at least seventeen times over a period of time, at least eighteen times over a period of time, at least nineteen times over a period of time, or at least twenty times over a period of time.
  • non-human animals comprising inactivated genes (e.g., Rag1, Rag2, Il2rg, or Fah genes or combination thereof) and/or humanized genes (e.g., IL6, OSM, GH, or combination thereof).
  • inactivated genes e.g., Rag1, Rag2, Il2rg, or Fah genes or combination thereof
  • humanized genes e.g., IL6, OSM, GH, or combination thereof.
  • various methods are provided for making a non-human animal comprising inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • various methods are provided for making a non-human animal comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • various methods are provided for making a non-human animal comprising inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • various methods are provided for making a non-human animal comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • inactivated non-human animal Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene (or any combination thereof) or for making a non-human animal genome or non-human animal cell comprising inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • various methods are provided for making inactivated non-human animal Rag1, Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene (or any combination thereof) or for making a non-human animal genome or non-human animal cell comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • inactivated non-human animal Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene (or any combination thereof) or for making a non-human animal genome or non-human animal cell comprising inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • various methods are provided for making inactivated non-human animal Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene (or any combination thereof) or for making a non-human animal genome or non-human animal cell comprising inactivated Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • inactivated non-human animal Rag1, Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene (or any combination thereof) or for making a non-human animal genome or non-human animal cell comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene (or any combination thereof) as disclosed elsewhere herein.
  • Any convenient method or protocol for producing a genetically modified non-human animal e.g., mouse or rat
  • Any convenient method or protocol for producing a genetically modified organism is suitable for producing such a genetically modified non-human animal.
  • Such genetically modified non-human animals can be generated, for example, through gene knock-out at targeted Rag2, Il2rg, and Fah genes and gene humanization at a targeted IL6 gene and optionally a gene humanization at a targeted SIRPA gene.
  • Such genetically modified non-human animals can be generated, for example, through gene knock-out at targeted Rag1, Rag2, Il2rg, and Fah genes and gene humanization at a targeted IL6 gene and optionally a gene humanization at a targeted SIRPA gene.
  • a method of producing a non-human animal comprising inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene can comprise: (1) providing a pluripotent non-human animal cell (e.g., a non-human animal embryonic stem (ES) cell) comprising inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene; (2) introducing the genetically modified pluripotent non-human animal cell into a non-human animal host embryo; and (3) gestating (e.g., implanting and gestating) the non-human animal host embryo in a surrogate non-human animal mother.
  • a pluripotent non-human animal cell e.g., a non-human animal embryonic stem (ES) cell
  • ES non-human animal embryonic stem
  • a method of producing a non-human animal comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene can comprise: (1) providing a pluripotent non-human animal cell (e.g., a non-human animal embryonic stem (ES) cell) comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene; (2) introducing the genetically modified pluripotent non-human animal cell into a non-human animal host embryo; and (3) gestating (e.g., implanting and gestating) the non-human animal host embryo in a surrogate non-human animal mother.
  • a pluripotent non-human animal cell e.g., a non-human animal embryonic stem (ES) cell
  • ES non-human animal embryonic stem
  • a method of producing a non-human animal comprising inactivated Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene can comprise: (1) providing a pluripotent non-human animal cell (e.g., a non-human animal embryonic stem (ES) cell) comprising inactivated Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene; (2) introducing the genetically modified pluripotent non-human animal cell into a non-human animal host embryo; and (3) gestating (e.g., implanting and gestating) the non-human animal host embryo in a surrogate non-human animal mother.
  • a pluripotent non-human animal cell e.g., a non-human animal embryonic stem (ES) cell
  • ES non-human animal embryonic stem
  • a method of producing a non-human animal comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene can comprise: (1) providing a pluripotent non-human animal cell (e.g., a non-human animal embryonic stem (ES) cell) comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene; (2) introducing the genetically modified pluripotent non-human animal cell into a non-human animal host embryo; and (3) gestating (e.g., implanting and gestating) the non-human animal host embryo in a surrogate non-human animal mother.
  • a pluripotent non-human animal cell e.g., a non-human animal embryonic stem (ES) cell
  • ES non-human animal embryonic stem
  • a method of producing the non-human animals described elsewhere herein can comprise gestating (e.g., implanting and gestating) a non-human animal one-cell stage embryo comprising the inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene in a non-human animal surrogate mother.
  • a method of producing the non-human animals described elsewhere herein can comprise gestating (e.g., implanting and gestating) a non-human animal one-cell stage embryo comprising the inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene in a non-human animal surrogate mother.
  • a method of producing the non-human animals described elsewhere herein can comprise gestating (e.g., implanting and gestating) a non-human animal one-cell stage embryo comprising the inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene in a non-human animal surrogate mother.
  • a method of producing the non-human animals described elsewhere herein can comprise gestating (e.g., implanting and gestating) a non-human animal one-cell stage embryo comprising the inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene in a non-human animal surrogate mother.
  • a method of producing the non-human animals described elsewhere herein can comprise gestating (e.g., implanting and gestating) a non-human animal one-cell stage embryo comprising the inactivated Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene in a non-human animal surrogate mother.
  • a method of producing the non-human animals described elsewhere herein can comprise gestating (e.g., implanting and gestating) a non-human animal one-cell stage embryo comprising the inactivated Rag1, Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene in a non-human animal surrogate mother.
  • the method of producing a non-human animal comprising inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene can comprise: (1) modifying the genome of a non-human animal pluripotent cell (e.g., a non-human animal ES cell) to comprise the inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene; (2) identifying or selecting the genetically modified non-human animal pluripotent cell comprising the inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene; (3) introducing the genetically modified non-human animal pluripotent cell into a non-human animal host embryo; and (4) gestating (e.g., implanting and gestating) the non-human animal host embryo in a non-human animal surrogate mother.
  • a non-human animal pluripotent cell e.
  • the host embryo comprising modified non-human animal pluripotent cell e.g., a non-human animal ES cell
  • modified non-human animal pluripotent cell e.g., a non-human animal ES cell
  • the non-human animal surrogate mother can then produce an F0 generation non-human animal comprising the inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene (and capable of transmitting the genetic modifications through the germline).
  • the step of modifying the genome to comprise the inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene can be done in any background.
  • the modifying step can be done in a non-human animal pluripotent cell in which none of the Rag2, Il2rg, and Fah genes have already been inactivated and the IL6 gene has not been humanized and the SIRPA gene has not been humanized.
  • the modifying step for any one of the genes can be done in a non-human animal pluripotent cell in which one or more of Rag2, Il2rg, and Fah loci or genes have already been inactivated (e.g., one, two, or all three of the other loci or genes have already been inactivated) and/or the IL6 gene has already been humanized (and optionally the SIRPA gene has already been humanized). If two or more of the genes are inactivated in non-human animal pluripotent cells in which none of the other genes have already been inactivated/humanized, non-human animals comprising the separate inactivated/humanized genes can be crossed to produce non-human animals having the two or more inactivated/humanized genes.
  • the method of producing a non-human animal comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene can comprise: (1) modifying the genome of a non-human animal pluripotent cell (e.g., a non-human animal ES cell) to comprise the inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene; (2) identifying or selecting the genetically modified non-human animal pluripotent cell comprising the inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene; (3) introducing the genetically modified non-human animal pluripotent cell into a non-human animal host embryo; and (4) gestating (e.g., implanting and gestating) the non-human animal host embryo in a non-human animal surrogate mother.
  • the host embryo comprising modified non-human animal pluripotent cell e.g., a non-human animal ES cell
  • modified non-human animal pluripotent cell e.g., a non-human animal ES cell
  • the non-human animal surrogate mother can then produce an F0 generation non-human animal comprising the inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene (and capable of transmitting the genetic modifications through the germline).
  • the modifying step for any one of the genes can be done in a non-human animal pluripotent cell in which one or more of Rag1, Rag2, Il2rg, and Fah loci or genes have already been inactivated (e.g., one, two, or all three of the other loci or genes have already been inactivated) and/or the IL6 gene has already been humanized (and optionally the SIRPA gene has already been humanized). If two or more of the genes are inactivated in non-human animal pluripotent cells in which none of the other genes have already been inactivated/humanized, non-human animals comprising the separate inactivated/humanized genes can be crossed to produce non-human animals having the two or more inactivated/humanized genes.
  • the method of producing a non-human animal comprising inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene can comprise: (1) modifying the genome of a non-human animal pluripotent cell (e.g., a non-human animal ES cell) to comprise the inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene; (2) identifying or selecting the genetically modified non-human animal pluripotent cell comprising the inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene; (3) introducing the genetically modified non-human animal pluripotent cell into a non-human animal host embryo; and (4) gestating (e.g., implanting and gestating) the non-human animal host embryo in a non-human animal surrogate mother.
  • a non-human animal pluripotent cell e.g.,
  • the host embryo comprising modified non-human animal pluripotent cell e.g., a non-human animal ES cell
  • modified non-human animal pluripotent cell e.g., a non-human animal ES cell
  • the non-human animal surrogate mother can then produce an F0 generation non-human animal comprising the inactivated Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene (and capable of transmitting the genetic modifications through the germline).
  • the host embryo comprising modified non-human animal pluripotent cell e.g., a non-human animal ES cell
  • modified non-human animal pluripotent cell e.g., a non-human animal ES cell
  • the non-human animal surrogate mother can then produce an F0 generation non-human animal comprising the inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene (and capable of transmitting the genetic modifications through the germline).
  • the step of modifying the genome to comprise the inactivated Rag1, Rag2, Il2rg, and Fah genes and a humanized OSM gene and optionally a humanized SIRPA gene can be done in any background.
  • the modifying step can be done in a non-human animal pluripotent cell in which none of the Rag1, Rag2, Il2rg, and Fah genes have already been inactivated and the OSM gene has not been humanized and the SIRPA gene has not been humanized.
  • the modifying step for any one of the genes can be done in a non-human animal pluripotent cell in which one or more of Rag1, Rag2, Il2rg, and Fah loci or genes have already been inactivated (e.g., one, two, or all three of the other loci or genes have already been inactivated) and/or the OSM gene has already been humanized (and optionally the SIRPA gene has already been humanized). If two or more of the genes are inactivated in non-human animal pluripotent cells in which none of the other genes have already been inactivated/humanized, non-human animals comprising the separate inactivated/humanized genes can be crossed to produce non-human animals having the two or more inactivated/humanized genes.
  • the method of producing a non-human animal comprising inactivated Rag1, Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene can comprise: (1) modifying the genome of a non-human animal pluripotent cell (e.g., a non-human animal ES cell) to comprise the inactivated Rag1, Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene; (2) identifying or selecting the genetically modified non-human animal pluripotent cell comprising the inactivated Rag1, Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally a humanized SIRPA gene; (3) introducing the genetically modified non-human animal pluripotent cell into a non-human animal host embryo; and (4) gestating (e.g., implanting and gestating) the non-human animal host embryo in a non-human animal surrogate
  • the step of modifying the genome to comprise the inactivated Rag2, Il2rg, and Fah genes and a humanized IL6 gene and optionally a humanized SIRPA gene can be done in any background.
  • the modifying step can be done in a non-human animal one-cell stage embryo in which none of the Rag2, Il2rg, and Fah genes have already been inactivated and the IL6 gene has not been humanized (and optionally the SIRPA gene has not been humanized).
  • the modifying step for any one of the genes can be done in a non-human animal one-cell stage embryo in which one or more of Rag1, Rag2, Il2rg, and Fah loci or genes have already been inactivated (e.g., one, two, or all three of the other loci or genes have already been inactivated) and/or the IL6 gene has already been humanized (and optionally the SIRPA gene has already been humanized). If two or more of the genes are inactivated in non-human animal one-cell stage embryos in which none of the other genes have already been inactivated/humanized, non-human animals comprising the separate inactivated/humanized genes can be crossed to produce non-human animals having the two or more inactivated/humanized genes.
  • the modifying step for any one of the genes can be done in a non-human animal one-cell stage embryo in which one or more of Rag2, Il2rg, and Fah loci or genes have already been inactivated (e.g., one, two, or all three of the other loci or genes have already been inactivated) and/or one or both of the IL6 gene and the OSM gene has already been humanized (and optionally the SIRPA gene has already been humanized).
  • non-human animals comprising the separate inactivated/humanized genes can be crossed to produce non-human animals having the two or more inactivated/humanized genes.
  • the genes can be inactivated/humanized by any known means (e.g., through use of a nuclease agent, through use of an exogenous donor nucleic acid, or through use of both a nuclease agent and an exogenous donor nucleic acid).
  • a nuclease agent e.g., through use of a nuclease agent, through use of an exogenous donor nucleic acid, or through use of both a nuclease agent and an exogenous donor nucleic acid.
  • two or more or all of the Rag2, Il2rg, and Fah genes can be inactivated simultaneously via multiplex genome editing.
  • two or more or all of the Rag2, Il2rg, and Fah genes can be inactivated and the IL6 gene and the OSM gene and optionally the SIRPA gene can be humanized simultaneously or sequentially in different cells to produce different non-human animals that are subsequently crossed to produce non-human animals with all of the inactivated/humanized genes (e.g., Rag2 can be inactivated in a first cell to produce a first non-human animal, and Il2rg can be inactivated in a second cell to produce a second non-human animal that is crossed to the first non-human animal).
  • two or more or all of the Rag2, Il2rg, and Fah genes can be inactivated and the IL6 and OSM genes and optionally the SIRPA gene can be humanized sequentially in the same cell.
  • two or more or all of the Rag1, Rag2, Il2rg, and Fah genes can be inactivated simultaneously via multiplex genome editing (e.g., Rag1 and Rag2 can be inactivated simultaneously in the same cell).
  • nucleases examples include a Transcription Activator-Like Effector Nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, and Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems (e.g., CRISPR/Cas9 systems) or components of such systems (e.g., CRISPR/Cas9).
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeats
  • Cas CRISPR-associated systems
  • the nuclease comprises a Cas9 protein and a guide RNA.
  • a nucleic acid insert in the endogenous Rag1 gene, Rag2 gene, Rag1 and Rag2 genes, Il2rg gene, Fah gene, or IL6 gene can result in addition of a nucleic acid sequence of interest in the endogenous Rag1 gene, Rag2 gene, Rag1 and Rag2 genes, Il2rg gene, Fah gene, or IL6 gene (or optionally SIRPA gene), deletion of a nucleic acid sequence of interest in the endogenous Rag1 gene, Rag2 gene, Rag1 and Rag2 genes, Il2rg gene, Fah gene, or IL6 gene (or optionally SIRPA gene), or replacement of a nucleic acid sequence of interest in the endogenous Rag1 gene, Rag2 gene, Rag1 and Rag2 genes, Il2rg gene, Fah gene, or IL6 gene (or optionally SIRPA gene) (i.e., deleting a segment of the endogenous Rag1 gene, Rag2 gene, Rag1 and Rag2 genes, Il2rg gene, Fah gene, or
  • FISH fluorescence-mediated in situ hybridization
  • comparative genomic hybridization isothermic DNA amplification
  • quantitative hybridization to an immobilized probe(s) include INVADER® Probes, TAQMAN® Molecular Beacon probes, or ECLIPSETM probe technology (see, e.g., US 2005/0144655, incorporated herein by reference in its entirety for all purposes).
  • the various methods provided herein allow for the generation of a genetically modified F0 non-human animal wherein the cells of the genetically modified F0 non-human animal comprise the inactivated Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally humanized SIRPA gene (or any combination thereof).
  • the cells of the genetically modified F0 non-human animal can be heterozygous for the inactivated Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally humanized SIRPA gene (or any combination thereof) or can be homozygous for the inactivated Rag2, Il2rg, and Fah genes and humanized IL6 and OSM genes and optionally humanized SIRPA gene (or any combination thereof).
  • any of the methods described herein can also be used, for example, for making non-human animals comprising a humanized Growth Hormone gene or optionally further comprising a humanized Growth Hormone gene in combination with any of the other inactivated genes and/or humanized genes disclosed herein.
  • treatment with mouse or rat IL-6 can induce Signal Transducer and Activator of Transcription 3 (STAT3) phosphorylation in both primary murine and rat hepatocytes, but not in human hepatocytes, consistent with suboptimal interaction between mouse or rat IL-6 ligand and human IL-6R.
  • STAT3 Signal Transducer and Activator of Transcription 3
  • mIL-6R expression in humanized livers was detected by immunoreactivity to an antibody ( FIG. 8 A ) or in situ hybridization to a FLAG-specific miRNAscope probe that recognizes a FLAG-tag sequence fused to the mIL-6R transgene ( FIG. 2 C ). Since no selection procedure was applied before transplantation, humanized livers consisted of both mIL-6R expressing (FLAG-positive) and non-expressing (FLAG-negative) regions of FAH-expressing (hASGR1-expressing) human hepatocytes.
  • GP130 186-Y190del expression was detected by in situ hybridization to a RNAscope probe specific to a GFP tag sequence in the transgene.
  • Humanized livers consisted of both mIL-6R expressing (GFP-positive) and non-expressing (GFP-negative) regions of hASGR1-expressing human hepatocytes ( FIG. 2 G ).
  • GFP-positive, GP130 Y186-Y190del expressing areas showed a complete disappearance of lipid droplet accumulation ( FIG. 2 G ). Therefore, GP130 activation is sufficient to protect humanized engraftments from hepatic steatosis in humanized liver mice.
  • huIL-6 expression was maintained in the serum of humanized liver mice until 8 weeks post-transplantation, when tissues were harvested.
  • AAV-huIL-6 dosed FSRG mice expressed 1.74.8 ng/mL of huIL-6 in the serum and exhibited robust phosphorylation of STAT3 in the liver ( FIG. 3 A ).
  • neither serum hIL-6, nor liver STAT3 phosphorylation were detectable in control humanized liver mice.
  • humanized livers of mice expressing with huIL-6 showed a substantial decrease of eosin-negative area ( ⁇ 2-6-fold, almost to baseline level), mostly in FAH+ regions, indicative of a reduction of lipid droplet accumulation ( FIG. 3 B ) and supporting a protective role of huIL-6 against hepatic steatosis in this model.
  • Lipopolysaccharide (LPS) treatment resulted in induction of huIL-6 expression in the serum of mice with homozygous humanized IL-6 allele ( FIG. 4 A ).
  • LPS Lipopolysaccharide
  • FIG. 4 A Lipopolysaccharide
  • huIL-6 treatment can reverse the steatosis phenotype in humanized livers. This question was addressed in both humanized liver mouse and rat models. Rather than administering AAV9-huIL-6 before hepatocyte transplantation, viral dosing was performed in FSRG mice or FRG rats that were already engrafted with human hepatocytes. At 4 weeks after AAV9-huIL-6 dosing, tissues were collected. Again, huIL-6 expression and hepatic IL-6 pathway activation was confirmed by serum huIL-6 and human CRP, liver pSTAT3, and qRT-PCR for IL-6 target genes ( FIGS. 11 A- 11 C and FIGS. 12 A- 12 C ).
  • huIL-6 dosing led to a nearly complete elimination of lipid droplet accumulation, marked by robust reduction of eosin-negative vacuoles in FAH+ regions and disappearance of Oil-Red-O staining in humanized livers.
  • activated IL-6R signaling in human hepatocytes not only prevents, but also reverses, hepatic steatosis formation in humanized liver rodents.
  • dosing with hOSM, another hepatic GP130/STAT3 pathway activating ligand also led to correction of liver fattiness in humanized liver mice ( FIGS. 14 A- 14 D ), supporting a protective role of GP130 pathway against lipid droplet accumulation in livers.
  • Kupffer cells are one of the major sources of IL-6 in livers. These cells are located adjacent to hepatocytes where Kupffer cell-derived IL-6 could support IL-6R pathway activation in nearby hepatocytes without elevating circulating IL-6 to harmful levels. We therefore attempted to address the role of Kupffer cells in protecting human hepatocytes from lipid droplet accumulation. To this end, we performed human immune cell reconstitution in FSRG mice prior to human hepatocyte transplantation ( FIG. 6 A ). Human fetal liver cells containing hematopoietic stem cells were engrafted into irradiated neonatal FSRG pups.
  • CSF1R colony-stimulating factor 1 receptor pathway
  • FIG. 6 E Immunohistochemical analysis confirmed a more than 3-fold increase in lipid droplet accumulation, marked by eosin-negative vacuoles in FAH-positive, humanized regions of the liver ( FIG. 6 E ). Therefore, Kupffer cells producing human IL-6 play a major role in controlling lipid droplet accumulation in hepatocytes in this model.
  • Hepatic steatosis in humanized liver rodent models resulting from incompatibility between human hepatocyte-expressed receptors and host-derived ligands, provides an opportunity to identify important signaling pathways involved in fatty liver disease.
  • excessive lipid droplet accumulation in humanized livers is associated with incompatibility between rodent IL6 ligand expressed by non-parenchymal cells of host animals and human IL6R expressed on donor hepatocytes.
  • lipid accumulation could be corrected by ectopic expression of rodent IL6R or constitutive activation of GP130 in donor hepatocytes.
  • Lipid droplet accumulation in human engraftment represents a major defect in humanized liver models that greatly compromises their accuracy in recapitulating normal human liver biology.
  • Our observations show an important role of both Kupffer cell-derived IL6 and its downstream IL6R/GP130 signaling in regulating lipid accumulation in hepatocytes and provide methods to improve humanized liver animal models.
  • the FSRG mouse strain was developed with Regeneron VELOCIGENE technology and was rendered immune-deficient by deletion of Rag2 and IL-2Rg genes.
  • the SIRPa gene was also humanized to allow for superior engraftment of human tissue (prevents phagocytosis of incoming human cells by facilitating “don't-eat-me-signal” to murine phagocytes).
  • FAH was deleted to induce murine liver ablation and allow for human hepatocyte engraftment.
  • the FRG rat strain is described in Carbonaro et al., “Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model,” Sci. Rep.
  • NTBC 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione
  • Hepatocyte number and viability was determined by ViaStain AOPI Staining Solution (Nexcelom CS2-0106), using the Nexcelom Cellometer Auto 2000.
  • 0.5-1 million live hepatocytes in 100 ⁇ L Roswell Park Memorial Institute (RPMI) 1640 Medium were injected into the spleen via a 32 gauge needle.
  • NTBC was withdrawn from the drinking water the day before transplant and mice were put into an NTBC on/off cycling schedule, as follows: 7 days off, 3 days on for the first 2.5-3 weeks, then gradually increasing the NTBC off times to 8, 10, 14, then 28 days between 3-day on cycles.
  • HSC Engraftment and Immune Check Single cell suspension of human fetal liver tissue (FL) was prepared by Collagenase D digestion (100 ng/mL; Roche) for 25 minutes at 37° C.
  • Human CD34+ hematopoietic stem cells (HSCs) were isolated from the cell suspension by positive immunomagnetic selection using anti-human CD34 microbeads according to the manufacturer's instructions (Miltenyi Biotec). Newborn pups were sublethally irradiated (360 cGy; X-RAD 320 irradiator) 4-24 hours prior to an intrahepatic injection of 1 ⁇ 10 5 human FL-derived CD34+ cells.
  • Engraftment with human immune system was checked by retro-orbital bleed of mice 12 weeks post-HSC injection and red blood cell lysed blood cells were analyzed by FACS for human CD45 and mouse CD45. Gating was performed on hCD45+/mCD45 ⁇ cells and human CD45+ population was analyzed for CD3 (T cells), CD19 (B cells), NKp46 (NK cells), and CD14 (monocytes/macrophages). Upon engraftment check, mice were subjected to human hepatocyte transplantation as further described.
  • Hepatocytes were treated with recombinant human IL-6 (ab119444), mouse IL-6 (ab238300) or rat IL-6 (Cell Applications, RP3009), human HGF (R&D 294-HG-025), or mouse HGF (R&D 2207-HG-025) for 15 minutes at 50 ng/mL.
  • Cells were lysed using RIPA buffer (ThermoFisher, 89900), containing protease and phosphatase inhibitors (ThermoFisher, A32965 & 88667), run by SDS-PAGE and probed using antibodies against pSTAT3, total STAT3 (Cell Signaling 9145, 4904), and B-actin (Sigma A5316).
  • Adeno-associated Virus (AAV) Production and Delivery. Recombinant AAV was produced by transient transfection of HEK 293T cells. Briefly, cells were transfected with AAV Rep-Cap, Adenovirus Helper, and AAV genome plasmids using PEI-Max (Polysciences). Virus containing supernatant were concentrated by tangential flow filtration and cells were lysed by sequential freeze and thaw (3 ⁇ ). Lysates were treated with Benzonase (Millipore Sigma) for one hour at 37° C. and clarified by centrifugation and filtration (0.2 ⁇ m PES).
  • AAV was purified from clarified cell lysates and concentrated supernatant by iodixanol gradient ultracentrifugation. Virus fractions were concentrated and buffer-exchanged to 1 ⁇ PBS+0.001% Pluronic F68 (Thermo Fisher Scientific) using Amicon 100 kDa MWCO Ultra Centrifugal filters (Millipore Sigma). AAV genomes were quantified by qPCR using TaqMan primers and probes specific for inverted terminal repeats. A standard curve was generated using serial dilutions of virus with a known concentration. Adeno-associated viruses (AAVs) were delivered intravenously by tail-vein injection at 5.00E+11 viral genomes (VG)/mouse.
  • AAVs Adeno-associated viruses
  • Lentiviral Vector Production, Titration and Infection Lentiviral particles were produced following standard lipofectamine-mediated co-transfection of HEK 293T cells with the transfer plasmid encoding mIL-6R and rIL-6R under the CMV promoter (pLVX-CMV-, subcloned from pLVX-EF1a-IRES-puro plasmid, Takara), a second generation packaging plasmid encoding the gag, pol and rev genes (psPAX2, obtained from the Tronolab at Indiana Polytechnique F ⁇ d ⁇ rale de Lausanne, Switzerland) and a plasmid encoding the vesicular stomatitis virus envelope glycoprotein G (VSV-G) as envelope plasmid (pMD2-G, Tronolab).
  • VSV-G vesicular stomatitis virus envelope glycoprotein G
  • the DNA mix was prepared by mixing 20 ⁇ g of transfer plasmid DNA, 20 ⁇ g of packaging plasmid and 10 ⁇ g of envelope plasmid, 1.5 mL of Opti-MEM with 60 ⁇ L of PLUS' Reagent (Life Technologies). In parallel 100 mL of Lipofectamine® TLX (Life Technologies) was diluted in 1.5 mL of OptiMEM medium. DNA mix was then added to the lipofectamine mix and the new combined solution was incubated at room temperature for 20 minutes before being added directly to the cells dropwise. The culture medium was changed 6 to 8 hours after transfection and the cells were then incubated for 48 hours at 37° C. in an incubator with 5% CO 2 atmosphere.
  • the resuspended virus was finally processed through a series of short centrifugations (30 seconds at 13500 rpm) to clarify the lentiviral solution of remaining debris.
  • the batches of lentiviral particles were titrated by RT-qPCR using a SYBR® technology-based kit from Clontech/Takara then stocked at ⁇ 80° C. until use for transduction.
  • Lentivirus infection of human hepatocytes was done ex-vivo prior to engraftment, at an MOI of 5.00E+04 VG/cell. Cells were infected for 30 minutes in suspension immediately upon thawing cryopreserved cells, washed with PBS, and prepared for engraftment as above.

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Family Cites Families (23)

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Publication number Priority date Publication date Assignee Title
WO1999005266A2 (fr) 1997-07-26 1999-02-04 Wisconsin Alumni Research Foundation Transfert de noyau entre des especes differentes
US20050144655A1 (en) 2000-10-31 2005-06-30 Economides Aris N. Methods of modifying eukaryotic cells
US6596541B2 (en) 2000-10-31 2003-07-22 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
US6586251B2 (en) 2000-10-31 2003-07-01 Regeneron Pharmaceuticals, Inc. Methods of modifying eukaryotic cells
AUPR451401A0 (en) 2001-04-20 2001-05-24 Monash University A method of nuclear transfer
US7612250B2 (en) 2002-07-29 2009-11-03 Trustees Of Tufts College Nuclear transfer embryo formation method
EP1802193B1 (fr) 2004-10-19 2014-04-30 Regeneron Pharmaceuticals, Inc. Methode de production d'une souris homozygote pour une modification genetique
CN101117633B (zh) 2006-08-03 2011-07-20 上海交通大学附属儿童医院 一种细胞核移植方法
KR101472519B1 (ko) 2007-06-05 2014-12-29 오레곤 헬스 앤드 사이언스 유니버시티 Ｉｎ ｖｉｖｏ에서 인간 간세포 증식 방법
JP2012525154A (ja) * 2009-05-01 2012-10-22 オレゴン ヘルス アンド サイエンス ユニバーシティ 生体内でヒト肝細胞を増大させる方法
MX368932B (es) 2009-06-26 2019-10-22 Regeneron Pharma Anticuerpos biespecificos facilmente aislados con formato de inmunoglobulina original.
JP5841322B2 (ja) 2010-04-22 2016-01-13 オレゴン ヘルス アンド サイエンス ユニバーシティ フマリルアセト酢酸ヒドロラーゼ（ｆａｈ）欠損性ブタおよびその使用
JP6484444B2 (ja) 2011-08-26 2019-03-13 イエキュリス コーポレーションＹｅｃｕｒｉｓ Ｃｏｒｐｏｒａｔｉｏｎ フマリルアセト酢酸ヒドロラーゼ（ｆａｈ）欠損及び免疫不全ラット、並びにそれらの使用
RU2751240C2 (ru) 2011-10-28 2021-07-12 Регенерон Фармасьютикалс, Инк. Гуманизированные il-6 и рецептор il-6
JP6275120B2 (ja) 2012-04-25 2018-02-07 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． 大きい標的化ベクターによるヌクレアーゼ媒介標的化
FI3597749T3 (fi) 2012-05-25 2023-10-09 Univ California Menetelmiä ja koostumuksia rna-ohjattua kohde-dna-modifikaatiota varten ja rna-ohjattua transkription modulaatiota varten
JP6475172B2 (ja) 2013-02-20 2019-02-27 リジェネロン・ファーマシューティカルズ・インコーポレイテッドＲｅｇｅｎｅｒｏｎ Ｐｈａｒｍａｃｅｕｔｉｃａｌｓ， Ｉｎｃ． ラットの遺伝子組換え
EP3456831B1 (fr) 2013-04-16 2021-07-14 Regeneron Pharmaceuticals, Inc. Modification ciblée du génome d'un rat
SMT202500280T1 (it) 2013-09-23 2025-09-12 Regeneron Pharma Animali non umani aventi un gene umanizzato della proteina regolatrice del segnale
MX388127B (es) 2013-12-11 2025-03-19 Regeneron Pharma Metodos y composiciones para la modificacion dirigida de un genoma.
MX384887B (es) 2014-06-23 2025-03-14 Regeneron Pharma Ensamblaje de adn mediado por nucleasa.
CA3176380A1 (fr) 2014-11-21 2016-05-26 Regeneron Pharmaceuticals, Inc. Procedes et compositions pour modification genetique ciblee utilisant des arn guides apparies
JP6860752B2 (ja) * 2018-12-14 2021-04-21 公益財団法人実験動物中央研究所 ヒト肝細胞が移植された非ヒト脊椎動物及びその製造方法

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